Viral material and nucleotide fragments associated with multiple sclerosis, for diagnostic, prophylactic and therapeutic purpose

ABSTRACT

Viral material, in the isolated or purified state, in which the genome comprises a nucleotide sequence chosen from the group including sequences SEQ ID NO:46, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 and SEQ ID NO:56, their complementary sequences and their equivalent sequences, in particular nucleotide sequences displaying, for any succession of 100 contiguous monomers, at least 50% and preferably at least 70% homology with the said sequences SEQ ID NO:46, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 and SEQ ID NO:56, respectively, and their complementary sequences.

[0001] Multiple sclerosis (MS) is a demyelinating disease of the centralnervous system (CNS) the cause of which remains as yet unknown.

[0002] Many studies have supported the hypothesis of a viral aetiologyof the disease, but none of the known viruses tested has proved to bethe causal agent sought: a review of the viruses sought for severalyears in MS has been compiled by E. Norrby (1) and R. T. Johnson (2).

[0003] Recently, a retrovirus different from the known humanretroviruses has been isolated in patients suffering from MS (3, 4, and5). The authors were also able to show that this retrovirus could betransmitted in vitro, that patients suffering from MS producedantibodies capable of recognizing proteins associated with the infectionof leptomeningeal cells by this retrovirus, and that the expression ofthe latter could be strongly stimulated by the immediate-early genes ofsome herpes-viruses (6).

[0004] All these results point to the role in MS of at least one unknownretrovirus or of a virus having reverse transcriptase activity which isdetectable according to the method published by H. Perron (3) andqualified as “LM7-like RT” activity. The content of the publicationidentified by (3) is incorporated in the present description byreference.

[0005] Recently, the Applicant's studies have enabled two continuouscell lines infected with natural isolates originating from two differentpatients suffering from MS to be obtained by a culture method asdescribed in the document WO-A-93/20188, the content of which isincorporated in the present description by reference. These two lines,derived from human choroid plexus cells, designated LM7PC and PLI-2,were deposited with the ECACC on Jul. 22, 1992 and Jan. 8, 1993,respectively, under numbers 92072201 and 93010817, in accordance withthe provisions of the Budapest Treaty. Moreover, the viral isolatespossessing LM7-like RT activity were also deposited with the ECACC underthe overall designation of “strains”. The “strain” or isolate harbouredby the PLI-2 line, designated POL-2, was deposited with the ECACC onJul. 22, 1992 under No. V92072202. The “strain” or isolate harboured bythe LM7PC line, designated MS7PG, was deposited with the ECACC on Jan.8, 1993 under No. V93010816.

[0006] Starting from the cultures and isolates mentioned above,characterized by biological and morphological criteria, the next stepwas to endeavour to characterize the nucleic acid material associatedwith the viral particles produced in these cultures.

[0007] The portions of the genome which have already been characterizedhave been used to develop tests for molecular detection of the viralgenome and immunoserological tests, using the amino acid sequencesencoded by the nucleotide sequences of the viral genome, in order todetect the immune response directed against epitopes associated with theinfection and/or viral expression.

[0008] These tools have already enabled an association to be confirmedbetween MS and the expression of the sequences identified in the patentscited later. However, the viral system discovered by the Applicant isrelated to a complex retroviral system. In effect, the sequences to befound encapsidated in the extracellular viral particles produced by thedifferent cultures of cells of patients suffering from MS show clearlythat there is coencapsidation of retroviral genomes which are relatedbut different from the “wild-type” retroviral genome which produces theinfective viral particles. This phenomenon has been observed betweenreplicative retroviruses and endogenous retroviruses belonging to thesame family, or even heterologous retroviruses. The notion of endogenousretroviruses is very important in the context of our discovery since, inthe case of MSRV-1, it has been observed that endogenous retroviralsequences comprising sequences homologous to the MSRV-1 genome exist innormal human DNA. The existence of endogenous retroviral elements (ERV)related to MSRV-1 by all or part of their genome explains the fact thatthe expression of the MSRV-1 retrovirus in human cells is able tointeract with closely related endogenous sequences. These interactionsare to be found in the case of pathogenic and/or infectious endogenousretroviruses (for example some ecotropic strains of the murine leukaemiavirus), and in the case of exogenous retroviruses whose nucleotidesequence may be found partially or wholly, in the form of ERVS, in thehost animal's genome (e.g. mouse exogenous mammary tumor virustransmitted via the milk). These interactions consist mainly of (i) atrans-activation or coactivation of ERVs by the replicative retrovirus(ii) and “illegitimate” encapsidation of RNAs related to ERVS, or ofERVs—or even of cellular RNAs—simply possessing compatible encapsidationsequences, in the retroviral particles produced by the expression of thereplicative strain, which are sometimes transmissible and sometimes witha pathogenicity of their own, and (iii) more or less substantialrecombinations between the coencapsidated genomes, in particular in thephases of reverse transcription, which lead to the formation of hybridgenomes, which are sometimes transmissible and sometimes with apathogenicity of their own.

[0009] Thus, (i) different sequences related to MSRV-1 have been foundin the purified viral particles; (ii) molecular analysis of thedifferent regions of the MSRV-1 retroviral genome should be carried outby systematically analyzing the coencapsidated, interfering and/orrecombined sequences which are generated by the infection and/orexpression of MSRV-1; furthermore, some clones may have defectivesequence portions produced by the retroviral replication and templateerrors and/or errors of transcription of the reverse transcriptase;(iii) the families of sequences related to the same retroviral genomicregion provide the means for an overall diagnostic detection which maybe optimized by the identification of invariable regions among theclones expressed, and by the identification of reading framesresponsible for the production. of antigenic and/or pathogenicpolypeptides which may be produced only by a portion, or even by justone, of the clones expressed, and, under these conditions, thesystematic analysis of the clones expressed in the region of a givengene enables the frequency of variation and/or of recombination of theMSRV-1 genome in this region to be evaluated and the optimal sequencesfor the applications, in particular diagnostic applications, to bedefined; (iv) the pathology caused by a retrovirus such as MSRV-1 may bea direct effect of its expression and of the proteins or peptidesproduced as a result thereof, but also an effect of the activation, theencapsidation or the recombination of related or heterologous genomesand of the proteins or peptides produced as a result thereof; thus,these genomes associated with the expression of and/or infection byMSRV-1 are an integral part of the potential pathogenicity of thisvirus, and hence constitute means of diagnostic detection and specialtherapeutic targets. Similarly, any agent associated with or cofactor ofthese interactions responsible for the pathogenesis in question, such asMSRV-2 or the glyotoxic factor which are described in the patentapplication published under No. FR-2,716,198, may participate in thedevelopment of an overall and very effective strategy for the diagnosis,prognosis, therapeutic monitoring and/or integrated therapy of MS inparticular, but also of any other disease associated with the sameagents.

[0010] In this context, a parallel discovery has been made in anotherautoimmune disease, rheumatoid arthritis (RA), which has been describedin the French Patent Application filed under No. 95/02960. Thisdiscovery shows that, by applying methodological approaches similar tothe ones which were used in the Applicant's work on MS, it was possibleto identify a retrovirus expressed in RA which shares the sequencesdescribed for MSRV-1 in MS, and also the coexistence of an associatedMSRV-2 sequence also described in MS. As regards MSRV-1, the sequencesdetected in common in MS and RA relate to the pol and gag genes. In thecurrent state of knowledge, it is possible to associate the gag and polsequences described with the MSRV-1 strains expressed in these twodiseases.

[0011] The present patent application relates to various results whichare additional to those already protected by the following French PatentApplications:

[0012] No. 92/04322 of 03.04.1992, published under No. 2,689,519;

[0013] No. 92/13447 of 03.11.1992, published under No. 2,689,521;

[0014] No. 92/13443 of 03.11.1992, published under No. 2,689,520;

[0015] No. 94/01529 of 04.02.1994, published under No. 2,715,936;

[0016] No. 94/01531 of 04.02.1994, published under No. 2,715,939;

[0017] No. 94/01530 of 04.02.1994, published under No. 2,715,936;

[0018] No. 94/01532 of 04.02.1994, published under No. 2,715,937;

[0019] No. 94/14322 of 24.11.1994, published under No. 2,727,428;

[0020] and No. 94/15810 of 23.12.1994; published under No. 2,728,585.

[0021] The present invention relates, in the first place, to a viralmaterial, in the isolated or purified state, which may be recognized orcharacterized in different ways:

[0022] its genome comprises a nucleotide sequence chosen from the groupincluding the sequences SEQ ID NO:46, SEQ ID NO:51, SEQ ID NO:52, SEQ IDNO:53, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ IDNO:61, SEQ ID NO:89, their complementary sequences and their equivalentsequences, in particular nucleotide sequences displaying, for anysuccession of 100 contiguous monomers, at least 50% and preferably atleast 70% homology with the said sequences SEQ ID NO:46, SEQ ID NO:51,SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:59,SEQ ID NO:60 SEQ ID NO:61, SEQ ID NO:89, respectively, and theircomplementary sequences;

[0023] the region of its genome comprising the env and pol genes and aportion of the gag gene, excluding the subregion having a sequenceidentical or equivalent to SEQ ID NO:1, codes for any polypeptidedisplaying, for any contiguous succession of at least 30 amino acids, atleast 50% and preferably at least 70% homology with a peptide sequenceencoded by any nucleotide sequence chosen from the group including SEQID NO:46, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:56, SEQ IDNO:58, SEQ ID NO:59, SEQ ID NO:60 SEQ ID NO:61 SEQ ID NO:89 and theircomplementary sequences;

[0024] the pol gene comprises a nucleotide sequence partially or totallyidentical or equivalent to SEQ ID NO:57, excluding SEQ ID NO:1.

[0025] the gag gene comprises a nucleotide sequence partially or totallyidentical or equivalent to SEQ ID NO:88.

[0026] As indicated above, according to the present invention, the viralmaterial as defined above is associated with MS. And as defined byreference to the pol or gag gene of MSRV-1, and more especially to thesequences SEQ ID NOS 51, 56, 57, 59, 60, 61, 88 and 89, this viralmaterial is associated with RA.

[0027] The present invention also relates to different nucleotidefragments each comprising a nucleotide sequence chosen from the groupincluding:

[0028] (a) all the genomic sequences, partial and total, of the pol geneof the MSRV-1 virus, except for the total sequence of the nucleotidefragment defined by SEQ ID NO:1;

[0029] (b) all the genomic sequences, partial and total, of the env geneof MSRV-1;

[0030] (c) all the partial genomic sequences of the gag gene of MSRV-1;

[0031] (d) all the genomic sequences overlapping the pol gene and theenv gene of the MSRV-1 virus, and overlapping the pol gene and the gaggene;

[0032] (e) all the sequences, partial and total, of a clone chosen fromthe group including the clones FBd3 (SEQ ID NO:46), t pol (SEQ IDNO:51), JLBc1 (SEQ ID NO:52), JLBc2 (SEQ ID NO:53) and GM3 (SEQ IDNO:56), FBd13 (SEQ ID NO:58), LB19 (SEQ ID NO:59), LTRGAG12 (SEQ IDNO:60), FP6 (SEQ ID NO:61), G+E+A (SEQ ID

[0033] NO:89), excluding any nucleotide sequence identical to or lyingwithin the sequence defined by SEQ ID NO:1;

[0034] (f) sequences complementary to the said genomic sequences;

[0035] (g) sequences equivalent to the said sequences (a) to (e), inparticular nucleotide sequences displaying, for any succession of 100contiguous monomers, at least 50% and preferably at least 70% homologywith the said sequences (a) to (d). provided that this nucleotidefragment does not comprise or consist of the sequence ERV-9 as describedin LA MANTIA et al. (18).

[0036] The term genomic sequences, partial or total, includes allsequences associated by coencapsidation or by coexpression, orrecombined sequences.

[0037] Preferably, such a fragment comprises:

[0038] either a nucleotide sequence identical to a partial or totalgenomic sequence of the pol gene of the MSRV-1 virus, except for thetotal sequence of the nucleotide fragment defined by SEQ ID NO:1, oridentical to any sequence equivalent to the said partial or totalgenomic sequence, in particular one which is homologous to the latter;

[0039] or a nucleotide sequence identical to a partial or total genomicsequence of the env gene of the MSRV-1 virus, or identical to anysequence complementary to the said nucleotide sequence, or identical toany sequence equivalent to the said nucleotide sequence, in particularone which is homologous to the latter.

[0040] In particular, the invention relates to a nucleotide fragmentcomprising a coding nucleotide sequence which is partially or totallyidentical to a nucleotide sequence chosen from the group including:

[0041] the nucleotide sequence defined by SEQ ID NO:40, SEQ ID NO:62 orSEQ ID NO:89;

[0042] sequences complementary to SEQ ID NO:40, SEQ ID NO:62 or SEQ IDNO:89;

[0043] sequences equivalent, and in particular homologous to SEQ IDNO:40, SEQ ID NO:62 or SEQ ID NO:89;

[0044] sequences coding for all or part of the peptide sequence definedby SEQ ID NO:39, SEQ ID NO:63 or SEQ ID NO:90;

[0045] sequences coding for all or part of a peptide sequenceequivalent, in particular homologous to SEQ ID NO:39, SEQ ID NO:63 orSEQ ID NO:90, which is capable of being recognized by sera of patientsinfected with the MSRV-1 virus, or in whom the MSRV-1 virus has beenreactivated.

[0046] The invention also relates to any nucleic acid probe fordetection of a pathogenic and/or infective agent associated with MS,which is capable of hybridizing specifically with any fragment such asis defined above, belonging or lying within the genome of the saidpathogenic agent. It relates, in addition, to any nucleic acid probe fordetection of a pathogenic and/or infective agent associated with RA,which is capable of hybridizing specifically with any fragment asdefined above by reference to the pol and gag genes, and especially withrespect to the sequences SEQ ID NOS 40, 51, 56, 59, 60, 61, 62, 89 andSEQ ID NOS 39, 63 and 90.

[0047] The invention also relates to a primer for the amplification bypolymerization of an RNA or a DNA of a viral material, comprising anucleotide sequence identical or equivalent to at least one portion ofthe nucleotide sequence of any fragment such as is defined above, inparticular a nucleotide sequence displaying, for any succession of 10contiguous monomers, at least 70% homology with at least the saidportion of the said fragment. Preferably, the nucleotide sequence ofsuch a primer is identical to any one of the sequences chosen from thegroup including SEQ ID NO:47 to SEQ ID NO:50, SEQ ID NO:55 and SEQ IDNO:64 SEQ ID NO:86.

[0048] Generally speaking the invention also encompasses any RNA or DNA,and in particular replication vector, comprising a genomic fragment ofthe viral material such as is defined above, or a nucleotide fragmentsuch as is defined above.

[0049] The invention also relates to the different peptides encoded byany open reading frame belonging to a nucleotide fragment such as isdefined above, in particular any polypeptide, for example anyoligopeptide forming or comprising an antigenic determinant recognizedby sera of patients infected with the MSRV-1 virus and/or in whom theMSRV-1 virus has been reactivated. Preferably, this polypeptide isantigenic, and is encoded by the open reading frame beginning, in the5′-3′ direction, at nucleotide 181 and ending at nucleotide 330 of SEQID NO:1.

[0050] In particular, the invention relates to an antigenic polypeptiderecognized by the sera of patients infected with the MSRV-1 virus,and/or in whom the MSRV-1 virus has been reactivated, whose peptidesequence is partially or totally identical or is equivalent to thesequence defined by SEQ ID NO:39, SEQ ID NO:63 and SEQ ID NO:87; such asequence is identical, for example, to any sequence chosen from thegroup including the sequences SEQ ID NO:41 to SEQ ID NO:44, SEQ ID NO:63and SEQ ID NO:87.

[0051] The present invention also proposes mono- or polyclonalantibodies directed against the MSRV-1 virus, which are obtained by theimmunological reaction of a human or animal body to an immunogenic agentconsisting of an antigenic polypeptide such as is defined above.

[0052] The invention next relates to:

[0053] reagents for detection of the MSRV- virus, or of an exposure tothe latter, comprising, as reactive substance, a peptide, in particularan antigenic peptide, such as is defined above, or an anti-ligand, inparticular an antibody to the said peptide;

[0054] all diagnostic, prophylactic or therapeutic compositionscomprising one or more peptides, in particular antigenic peptides, suchas are defined above, or one or more anti-ligands, in particularantibodies to the peptides, discussed above; such a composition ispreferably, and by way of example, a vaccine composition.

[0055] The invention also relates to any diagnostic, prophylactic ortherapeutic composition, in particular for inhibiting the expression ofat least one pathogenic and/or infective agent associated with MScomprising a nucleotide fragment such as is defined above or apolynucleotide, in particular oligonucleotide, whose sequence ispartially identical to that of the said fragment, except for that of thefragment having the nucleotide sequence SEQ ID NO:1. Likewise, itrelates to any diagnostic, prophylactic or therapeutic composition, inparticular for inhibiting the expression of at least one pathogenicand/or infective agent associated with RA, comprising a nucleotidefragment such as is defined above by reference to the pol and gag genes,and especially with respect to the sequences SEQ ID NOS 40, 51, 56, 59,60, 61, 62 and 89.

[0056] According to the invention, these same fragments orpolynucleotides, in particular oligonucleotides, may participate in allsuitable compositions for detecting, according to any suitable processor method, a pathological and/or infective agent associated with MS andwith RA, respectively, in a biological sample. In such a process, an RNAand/or a DNA presumed to belong or originating from the saidpathological and/or infective agent, and/or their complementary RNAand/or DNA, is/are brought into contact with such a composition.

[0057] The present invention also relates to any process for detectingthe presence or exposure to such a pathological and/or infective agent,in a biological sample, by bringing this sample into contact with apeptide, in particular an antigenic peptide such as is defined above, oran anti-ligand, in particular an antibody to this peptide, such as isdefined above.

[0058] In practice, and for example, a device for detection of theMSRV-1 virus comprises a reagent such as is defined above, supported bya solid support which is immunologically compatible with the reagent,and a means for bringing the biological sample, for example a sample ofblood or of cerebrospinal fluid, likely to contain anti-MSRV-1antibodies, into contact with this reagent under conditions permitting apossible immunological reaction, the foregoing items being accompaniedby means for detecting the immune complex formed with this reagent.

[0059] Lastly, the invention also relates to the detection ofanti-MSRV-1 antibodies in a biological sample, for example a sample ofblood or of cerebrospinal fluid, according to which this sample isbrought into contact with a reagent such as is defined above, consistingof an antibody, under conditions permitting their possible immunologicalreaction, and the presence of the immune complex thereby formed with thereagent is then detected.

[0060] Before describing the invention in detail, different terms usedin the description and the claims are now defined:

[0061] strain or isolate is understood to mean any infective and/orpathogenic biological fraction containing, for example, viruses and/orbacteria and/or parasites, generating pathogenic and/or antigenic power,harboured by a culture or a living host; as an example, a viral strainaccording to the above definition can contain a coinfective agent, forexample a pathogenic protist,

[0062] the term “MSRV” used in the present description denotes anypathogenic and/or infective agent associated with MS, in particular aviral species, the attenuated strains of the said viral species or thedefective-interfering particles or particles containing coencapsidatedgenomes, or alternatively genomes recombined with a portion of theMSRV-1 genome, derived from this species. Viruses, and especiallyviruses containing RNA, are known to have a variability resulting, inparticular, from relatively high rates of spontaneous mutation (7),which will be borne in mind below for defining the notion ofequivalence,

[0063] human virus is understood to mean a virus capable of infecting,or of being harboured by human beings,

[0064] in view of all the natural or induced variations and/orrecombination which may be encountered when implementing the presentinvention, the subjects of the latter, defined above and in the claims,have been expressed including the equivalents or derivatives of thedifferent biological materials defined below, in particular of thehomologous nucleotide or peptide sequences,

[0065] the variant of a virus or of a pathogenic and/or infective agentaccording to the invention comprises at least one antigen recognized byat least one antibody directed against at least one correspondingantigen of the said virus and/or said pathogenic and/or infective agent,and/or a genome any part of which is detected by at least onehybridization probe and/or at least one nucleotide amplification primerspecific for the said virus and/or pathogenic and/or infective agent,such as, for example, for the MSRV-1 virus, the primers and probeshaving a nucleotide sequence chosen from SEQ ID No. 20 to SEQ ID No. 24,SEQ ID No. 26, SEQ ID No. 16 to SEQ ID No. 19, SEQ ID No. 31 to SEQ IDNo. 33, SEQ ID No. 45, SEQ ID No. 47, SEQ ID No. 48, SEQ ID No. 49, SEQID No. 50, SEQ ID No. 45 and their complementary sequences, underparticular hybridization conditions well known to a person skilled inthe art,

[0066] according to the invention, a nucleotide fragment or anoligonucleotide or polynucleotide is an arrangement of monomers, or abiopolymer, characterized by the informational sequence of the naturalnucleic acids, which is capable of hybridizing with any other nucleotidefragment under predetermined conditions, it being possible for thearrangement to contain monomers of different chemical structures and tobe obtained from a molecule of natural nucleic acid and/or by geneticrecombination and/or by chemical synthesis; a nucleotide fragment may beidentical to a genomic fragment of the MSRV-1 virus discussed in thepresent invention, in particular a gene of this virus, for example polor env in the case of the said virus,

[0067] thus, a monomer can be a natural nucleotide of nucleic acid whoseconstituent elements are a sugar, a phosphate group and a nitrogenousbase; in RNA the sugar is ribose, in DNA the sugar is 2-deoxyribose;depending on whether the nucleic acid is DNA or RNA, the nitrogenousbase is chosen from adenine, guanine, uracil, cytosine and thymine; orthe nucleotide can be modified in at least one of the three constituentelements; as an example, the modification can occur in the bases,generating modified bases such as inosine, 5-methyldeoxy- cytidine,deoxyuridine, 5-(dimethylamino)deoxyuridine, 2,6-diaminopurine,5-bromodeoxyuridine and any other modified base promoting hybridization;in the sugar, the modification can consist of the replacement of atleast one deoxyribose by a polyamide (8), and in the phosphate group,the modification can consist of its replacement by esters chosen, inparticular, from diphosphate, alkyl- and arylphosphonate andphosphorothioate esters,

[0068] “informational sequence” is understood to mean any orderedsuccession of monomers whose chemical nature and order in a referencedirection constitute or otherwise an item of functional information ofthe same quality as that of the natural nucleic acids,

[0069] hybridization is understood to mean the process during which,under suitable working conditions, two nucleotide fragments havingsufficiently complementary sequences pair to form a complex structure,in particular double or triple, preferably in the form of a helix,

[0070] a probe comprises a nucleotide fragment synthesized chemically orobtained by digestion or enzymatic cleavage of a longer nucleotidefragment, comprising at least six monomers, advantageously from 10 to100 monomers and preferably 10 to 30 monomers, and possessing aspecificity of hybridization under particular conditions; preferably, aprobe possessing fewer than 10 monomers is not used alone, but is usedin the presence of other probes of equally short size or otherwise;under certain special conditions, it may be useful to use probes of sizegreater than 100 monomers; a probe may be used, in particular, fordiagnostic purposes, such molecules being, for example, capture and/ordetection probes,

[0071] the capture probe may be immobilized on a solid support by anysuitable means, that is to say directly or indirectly, for example bycovalent bonding or passive adsorption,

[0072] the detection probe may be labelled by means of a label chosen,in particular, from radioactive isotopes, enzymes chosen, in particular,from peroxidase and alkaline phosphatase and those capable ofhydrolysing a chromogenic, fluorogenic or luminescent substrate,chromophoric chemical compounds, chromogenic, fluorogenic or luminescentcompounds, nucleotide base analogues and biotin,

[0073] the probes used for diagnostic purposes of the invention may beemployed in all known hybridization techniques, and in particular thetechniques termed “DOT-BLOT” (9), “SOUTHERN BLOT” (10), “NORTHERN BLOT”,which is a technique identical to the “SOUTHERN BLOT” technique butwhich uses RNA as target, and the SANDWICH technique (11);advantageously, the SANDWICH technique is used in the present invention,comprising a specific capture probe and/or a specific detection probe,on the understanding that the capture probe and the detection probe mustpossess an at least partially different nucleotide sequence,

[0074] any probe according to the present invention can hybridize invivo or in vitro with RNA and/or with DNA in order to block thephenomena of replication, in particular translation and/ortranscription, and/or to degrade the said DNA and/or RNA,

[0075] a primer is a probe comprising at least six monomers, andadvantageously from 10 to 30 monomers, possessing a specificity ofhybridization under particular conditions for the initiation of anenzymatic polymerization, for example in an amplification technique suchas PCR (polymerase chain reaction), in an elongation process such assequencing, in a method of reverse transcription or the like,

[0076] two nucleotide or peptide sequences are termed equivalent orderived with respect to one another, or with respect to a referencesequence, if functionally the corresponding biopolymers can performsubstantially the same role, without being identical, as regards theapplication or use in question, or in the technique in which theyparticipate; two sequences are, in particular, equivalent if they areobtained as a result of natural variability, in particular spontaneousmutation of the species from which they have been identified, or inducedvariability, as are two homologous sequences, homology being definedbelow,

[0077] “variability” is understood to mean any spontaneous or inducedmodification of a sequence, in particular by substitution and/orinsertion and/or deletion of nucleotides and/or of nucleotide fragments,and/or extension and/or shortening of the sequence at one or both ends;an unnatural variability can result from the genetic engineeringtechniques used, for example the choice of synthesis primers, degenerateor otherwise, selected for amplifying a nucleic acid; this variabilitycan manifest itself in modifications of any starting sequence,considered as reference, and capable of being expressed by a degree ofhomology relative to the said reference sequence,

[0078] homology characterizes the degree of identity of two nucleotideor peptide fragments compared; it is measured by the percentage identitywhich is determined, in particular, by direct comparison of nucleotideor peptide sequences, relative to reference nucleotide or peptidesequences,

[0079] this percentage identity has been specifically determined for thenucleotide fragments, clones in particular, dealt with in the presentinvention, which are homologous to the fragments identified, for theMSRV-1 Virus, by SEQ ID No. 1 to No. 9, SEQ ID NO:46, SEQ ID NO:51 toSEQ ID NO:53, SEQ ID NO:40, SEQ ID NO:56 and SEQ ID NO:57, as well asfor the probes and primers homologous to the probes and primersidentified by SEQ ID NO:20 to SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:16to SEQ ID NO:19, SEQ ID NO:31 to SEQ ID NO:33, SEQ ID NO:45, SEQ IDNO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:55, SEQ IDNO:40, SEQ ID NO:56 and SEQ ID NO:57; as an example, the smallestpercentage identity observed between the different general consensussequences of nucleic acids obtained from fragments of MSRV-1 viral RNA,originating from the LM7PC and PLI-2 lines according to a protocoldetailed later, is 67% in the region described in FIG. 1,

[0080] any nucleotide fragment is termed equivalent or derived from areference fragment if it possesses a nucleotide sequence equivalent tothe sequence of the reference fragment; according to the abovedefinition, the following in particular are equivalent to a referencenucleotide fragment:

[0081] a) any fragment capable of hybridizing at least partially withthe complement of the reference fragment,

[0082] b) any fragment whose alignment with the reference fragmentresults in the demonstration of a larger number of identical contiguousbases than with any other fragment originating from another taxonomicgroup,

[0083] c) any fragment resulting, or capable of resulting, from thenatural variability of the species from which it is obtained,

[0084] d) any fragment capable of resulting from the genetic engineeringtechniques applied to the reference fragment,

[0085] e) any fragment containing at least eight contiguous nucleotidesencoding a peptide which is homologous or identical to the peptideencoded by the reference fragment,

[0086] f) any fragment which is different from the reference fragment byinsertion, deletion or substitution of at least one monomer, orextension or shortening at one or both of its ends; for example, anyfragment corresponding to the reference fragment flanked at one or bothof its ends by a nucleotide sequence not coding for a polypeptide,

[0087] polypeptide is understood to mean, in particular, any peptide ofat least two amino acids, in particular an oligopeptide or protein,extracted, separated or substantially isolated or. synthesized throughhuman intervention, in particular those obtained by chemical synthesisor by expression in a recombinant organism,

[0088] polypeptide partially encoded by a nucleotide fragment isunderstood to mean a polypeptide possessing at least three amino acidsencoded by at least nine contiguous monomers lying within the saidnucleotide fragment,

[0089] an amino acid is termed analogous to another amino acid whentheir respective physicochemical properties, such as polarity,hydrophobicity and/or basicity and/or acidity and/or neutrality aresubstantially the same; thus, a leucine is analogous to an isoleucine.

[0090] any polypeptide is termed equivalent or derived from a referencepolypeptide if the polypeptides compared have substantially the sameproperties, and in particular the same antigenic, immunological,enzymological and/or molecular recognition properties; the following inparticular are equivalent to a reference polypeptide:

[0091] a) any polypeptide possessing a sequence in which at least oneamino acid has been replaced by an analogous amino acid,

[0092] b) any polypeptide having an equivalent peptide sequence,obtained by natural or induced variation of the said referencepolypeptide and/or of the nucleotide fragment coding for the saidpolypeptide,

[0093] c) a mimotope of the said reference polypeptide,

[0094] d) any polypeptide in whose sequence one or more amino acids ofthe L series are replaced by an amino acid of the D series, and viceversa,

[0095] e) any polypeptide into whose sequence a modification of the sidechains of the amino acids has been introduced, such as, for example, anacetylation of the amine functions, a carboxylation of the thiolfunctions, an esterification of the carboxyl functions,

[0096] f) any polypeptide in whose sequence one or more peptide bondshave been modified, such as, for example, carba, retro, inverso,retro-inverso, reduced and methylenoxy bonds,

[0097] (g) any polypeptide at least one antigen of which is recognizedby an antibody directed against a reference polypeptide,

[0098] the percentage identity characterizing the homology of twopeptide fragments compared is, according to the present invention, atleast 50% and preferably at least 70%.

[0099] In view of the fact that a virus possessing reverse transcriptaseenzymatic activity may be genetically characterized equally well in RNAand in DNA form, both the viral DNA and RNA will be referred to forcharacterizing the sequences relating to a virus possessing such reversetranscriptase activity, termed MSRV-1 according to the presentdescription.

[0100] The expressions of order used in the present description and theclaims, such as “first nucleotide sequence”, are not adopted so as toexpress a particular order, but so as to define the invention moreclearly.

[0101] Detection of a substance or agent is understood below to meanboth an identification and a quantification, or a separation orisolation, of the said substance or said agent.

[0102] A better understanding of the invention will be gained on readingthe detailed description which follows, prepared with reference to theattached figures, in which:

[0103]FIG. 1 shows general consensus sequences of nucleic acids of theMSRV-1B clones amplified by the PCR technique in the “pol” regiondefined by Shih (12), from viral DNA originating from the LM7PC andPLI-2 lines, and identified under the references SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO: 5 and SEQ ID NO: 6, and the common consensus withamplification primers bearing the reference SEQ ID NO:7;

[0104]FIG. 2 gives the definition of a functional reading frame for eachMSRV-1B/“PCR pol” type family, the said families A to D being defined,respectively, by the nucleotide sequences SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5 and SEQ ID NO:6 described in FIG. 1;

[0105]FIG. 3 gives an example of consensus of the MSRV-2B sequences,identified by SEQ ID NO:11;

[0106]FIG. 4 is a representation of the reverse transcriptase (RT)activity in dpm (disintegrations per minute) in the sucrose fractionstaken from a purification gradient of the virions produced by the Blymphocytes in culture from a patient suffering from MS;

[0107]FIG. 5 gives, under the same experimental conditions as in FIG. 4,the assay of the reverse transcriptase activity in the culture of a Blymphocyte line obtained from a control free from MS;

[0108]FIG. 6 shows the nucleotide sequence of the clone PSJ17 (SEQ IDNO:9);

[0109]FIG. 7 shows the nucleotide sequence SEQ ID NO:8 of the clonedesignated M003-P004;

[0110]FIG. 8 shows the nucleotide sequence SEQ ID NO:2 of the cloneF11-1; the portion located between the two arrows in the region of theprimer corresponds to a variability imposed by the choice of primerwhich was used for the cloning of F11-1; in this same figure, thetranslation into amino acids is shown;

[0111]FIG. 9 shows the nucleotide sequence SEQ ID NO:1, and a possiblefunctional reading frame of SEQ ID NO:1 in terms of amino acids; on thissequence, the consensus sequences of the pol gene are underlined;

[0112]FIGS. 10 and 11 give the results of a PCR, in the form of aphotograph under ultraviolet light of an ethidium bromide-impregnatedagarose gel, of the amplification products obtained from the primersidentified by SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19;

[0113]FIG. 12 gives a representation in matrix form of the homologybetween SEQ ID NO:1 of MSRV-1 and that of an endogenous retrovirusdesignated HSERV9; this homology of at least 65% is demonstrated by acontinuous line, the absence of a line meaning a homology of less than65%;

[0114]FIG. 13 shows the nucleotide sequence SEQ ID NO:46 of the cloneFBd3;

[0115]FIG. 14 shows the sequence homology between the clone FBd3 and theHSERV-9 retrovirus;

[0116]FIG. 15 shows the nucleotide sequence SEQ ID NO:51 of the clone tpol;

[0117]FIGS. 16 and 17 show, respectively, the nucleotide sequences SEQID NO:52 and SEQ ID NO:53 of the clones JLBc1 and JLBc2, respectively;

[0118]FIG. 18 shows the sequence homology between the clone JLBc1 andthe clone FBd3;

[0119] and FIG. 19 the sequence homology between the clone JLBc2 and theclone FBd3;

[0120]FIG. 20 shows the sequence homology between the clones JLBc1 andJLBc2;

[0121]FIGS. 21 and 22 show the sequence homology between the HSERV-9retrovirus and the clones JLBc1 and JLBc2, respectively;

[0122]FIG. 23 shows the nucleotide sequence SEQ ID NO:56 of the cloneGM3;

[0123]FIG. 24 shows the sequence homology between the HSERV-9 retrovirusand the clone GM3;

[0124]FIG. 25 shows the localization of the different clones studied,relative to the genome of the known retrovirus ERV9;

[0125]FIG. 26 shows the position of the clones F11-1, M003-P004, MSRV-1Band PSJ17 in the region hereinafter designated MSRV-1 pol*;

[0126]FIG. 27, split into three successive FIGS. 27a, 27 b and 27 c,shows a possible reading frame covering the whole of the pol gene;

[0127]FIG. 28 shows, according to SEQ ID NO:40, the nucleotide sequencecoding for the peptide fragment POL2B, having the amino acid sequenceidentified by SEQ ID NO:39;

[0128]FIG. 29 shows the OD values (ELISA tests) at 492 nm obtained for29 sera of MS patients and 32 sera of healthy controls tested with ananti-IgG antibody;

[0129]FIG. 30 shows the OD values (ELISA tests) at 492 nm obtained for36 sera of MS patients and 42 sera of healthy controls tested with ananti-IgM antibody;

[0130] FIGS. 31 to 33 show the results obtained (relative intensity ofthe spots) for 43 overlapping octapeptides covering the amino acidsequence 61-110, according to the Spotscan technique, respectively witha pool of MS sera, with a pool of control sera and with the pool of MSsera after deduction of a background corresponding to the maximum signaldetected on at least one octapeptide with the control serum (intensity=1), on the understanding that these sera were diluted to {fraction(1/50)}. The bar at the far right-hand end represents a graphic scalestandard unrelated to the serological test;

[0131]FIG. 34 shows the SEQ ID NO:41 and SEQ ID NO:42 of twopolypeptides comprising immunodominant [lacuna], while SEQ ID NO:43 and44 represent immunoreactive polypeptides specific to MS;

[0132]FIG. 35 shows the nucleotide sequence SEQ ID NO:59 of the cloneLB19 and three potential reading frames of SEQ ID NO:59 in terms ofamino acids;

[0133]FIG. 36 shows the nucleotide sequence SEQ ID NO:88 (GAG*) and apotential reading frame of SEQ ID NO:88 in terms of amino acids;

[0134]FIG. 37 shows the sequence homology between the clone FBd13 andthe HSERV-9 retrovirus; according to this representation, the continuousline means a percentage homology greater than or equal to 70% and theabsence of a line means a smaller percentage homology;

[0135]FIG. 38 shows the nucleotide sequence SEQ ID NO:61 of the cloneFP6 and three potential reading frames of SEQ ID NO:61 in terms of aminoacids;

[0136]FIG. 39 shows the nucleotide sequence SEQ ID NO:89 of the cloneG+E+A and three potential reading frames of SEQ ID NO:89 in terms ofamino acids;

[0137]FIG. 40, shows a reading frame found in the region E and codingfor an MSRV-1 retroviral protease identified by SEQ ID NO:90;

[0138]FIG. 41 shows the response of each serum of patients sufferingfrom MS, indicated by the symbol. (+), and of healthy patients,symbolised by (−), tested with an anti-IgG antibody, expressed as netoptical density at 492 nm;

[0139]FIG. 42 shows the response of each serum of patients sufferingfrom MS, indicated by the symbols (+) and (QS), and of healthy patients(−), tested with an anti-IgM antibody, expressed as net optical densityat 492 nm.

EXAMPLE 1

[0140] Obtaining Clones Designated MSRV-1B and MSRV-2B, Defining,Respectively, a Retrovirus MSRV-1 and a Coinfective Agent MSRV2, by“Nested” PCR Amplification of the Conserved POL Regions of Retroviruseson Virion Preparations Originating from the LM7PC and PLI-2 Lines

[0141] A PCR technique derived from the technique published by Shih (12)was used. This technique enables all trace of contaminant DNA to beremoved by treating all the components of the reaction medium withDNase. It concomitantly makes it possible, by the use of different butoverlapping primers in two successive series of PCR amplificationcycles, to increase the chances of amplifying a cDNA synthesized from anamount of RNA which is small at the outset and further reduced in thesample by the spurious action of the DNAse on the RNA. In effect, theDNase is used under conditions of activity in excess which enable alltrace of contaminant DNA to be removed before inactivation of thisenzyme remaining in the sample by heating to 85° C. for 10 minutes. Thisvariant of the PCR technique described by Shih (12) was used on a cDNAsynthesized from the nucleic acids of fractions of infective particlespurified on a sucrose gradient according to the technique described byH. Perron (13) from the “POL-2” isolate (ECACC No. V92072202) producedby the PLI-2 line (ECACC No. 92072201) on the one hand, and from theMS7PG isolate (ECACC No. V93010816) produced by the LM7PC line (ECACCNo. 93010817) on the other hand. These cultures were obtained accordingto the methods which formed the subject of the patent applicationspublished under Nos WO 93/20188 and WO 93/20189.

[0142] After cloning the products amplified by this technique with theTA Cloning Kit® and analysis of the sequence using an Applied Biosystemsmodel 373A Automatic Sequencer, the sequences were analysed using tneGeneworks® software on the latest available version of the Genebank®data bank.

[0143] The sequences cloned and sequenced from these samples correspond,in particular, to two types of sequence: a first type of sequence, to befound in the majority of the clones (55% of the clones originating fromthe POL-2 isolates of the PLI-2 culture, and 67% of the clonesoriginating from the MS7PG isolates of the LM7PC cultures), whichcorresponds to a family of “pol” sequences closely similar to, butdifferent from, the endogenous human retrovirus designated ERV-9 orHSERV-9, and a second type of sequence which corresponds to sequencesvery strongly homologous to a sequence attributed to another infectiveand/or pathogenic agent designated MSRV-2.

[0144] The first type of sequence, representing the majority of theclones, consists of sequences whose variability enables four subfamiliesof sequences to be defined. These subfamilies are sufficiently similarto one another for it to be possible to consider them to bequasi-species originating from the same retrovirus, as is well known forthe HIV-1 retrovirus (14), or to be the outcome of interference withseveral endogenous proviruses coregulated in the producing cells. Thesemore or less defective endogenous elements are sensitive to the sameregulatory signals possibly generated by a replicative provirus, sincethey belong to the same family of endogenous retroviruses (15). This newfamily of endogenous retroviruses, or alternatively this new retroviralspecies from which the generation of quasi-species has been obtained inculture, and which contains a consensus of the sequences describedbelow, is desig nated MSRV-1B.

[0145]FIG. 1 presents the general consensus sequences of the sequencesof the different MSRV-1B clones sequenced in this experiment, thesesequences being identified, respectively, by SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5 and SEQ ID NO:6. These sequences display a homology withrespect to nucleic acids ranging from 70% to 88% with the HSERV9sequence referenced X57147 and M37638 in the Genebankr data base. Four“consensus” nucleic acid sequences representative of differentquasi-species of a possibly exogenous retrovirus MSRV-1B, or ofdifferent subfamilies of an endogenous retrovirus MSRV-1B, have beendefined. These representative consensus sequences are presented in FIG.2, with the translation into amino acids. A functional reading frameexists for each subfamily of these MSRV-1B sequences, and it can be seenthat the functional open reading frame corresponds in each instance tothe amino acid sequence appearing on the second line under the nucleicacid sequence. The general consensus of the MSRV-1B sequence, identifiedby SEQ ID NO:7 and obtained by this PCR technique in the “pol” region,is presented in FIG. 1.

[0146] The second type of sequence representing the majority of theclones sequenced is represented by the sequence MSRV-2B presented inFIG. 3 and identified by SEQ ID NO:11. The differences observed in thesequences corresponding to the PCR primers are explained by the use ofdegenerate primers in mixture form used under different technicalconditions.

[0147] The MSRV-2B sequence (SEQ ID NO:11) is sufficiently divergentfrom the retroviral sequences already described in the data banks for itto be suggested that the sequence region in question belongs to a newinfective agent, designated MSRV-2. This infective agent would, inprinciple, on the basis of the analysis of the first sequences obtained,be related to a retrovirus but, in view of the technique used forobtaining this sequence, it could also be a DNA virus whose genome codesfor an enzyme which incidentally possesses reverse transcriptaseactivity, as is the case, for example, with the hepatitis B virus, HBV(12). Furthermore, the random nature of the degenerate primers used forthis PCR amplification technique may very well have permitted, as aresult of unforeseen sequence homologies or of conserved sites in thegene for a related enzyme, the amplification of a nucleic acidoriginating from a prokaryotic or eukaryotic pathogenic and/orcoinfective agent (protist).

EXAMPLE 2

[0148] Obtaining Clones Designated MSRV-1B and MSRV-2B, Defining aFamily MSRV-1 and MSRV2, by “Nested” PCR Amplification of the ConservedPOL Regions of Retroviruses on Preparations of B Lymphocytes from a NewCase of MS

[0149] The same PCR technique, modified according to the technique ofShih (12), was used to amplify and sequence the RNA nucleic acidmaterial present in a purified fraction of virions at the peak of“LM7-like” reverse transcriptase activity on a sucrose gradientaccording to the technique described by H. Perron (13), and according tothe protocols mentioned in Example 1, from a spontaneous lymphoblastoidline obtained by self-immortalization in culture of B lymphocytes froman MS patient who was seropositive for the Epstein-Barr virus (EBV),after setting up the blood lymphoid cells in culture in a suitableculture medium containing a suitable concentration of cyclosporin A. Arepresentation of the reverse transcriptase activity in the sucrosefractions taken from a purification gradient of the virions produced bythis line is presented in FIG. 4. Similarly, the culture supernatants ofa B line obtained under the same conditions from a control free from MSwere treated under the same conditions, and the assay of reversetranscriptase activity in the sucrose gradient fractions proved negativethroughout (background), and is presented in FIG. 5. Fraction 3 of thegradient corresponding to the MS B line and the same fraction withoutreverse transcriptase activity of the non-MS control gradient wereanalysed by the same RT-PCR technique as before, derived from Shih (12),followed by the same steps of cloning and sequencing as described inExample 1.

[0150] It is particularly noteworthy that the MSRV-1 and MSRV-2 typesequences are to be found only in the material associated with a peak of“LM7-like” reverse transcriptase activity originating from the MS Blymphoblastoid line. These sequences were not to be found with thematerial from the control (non-MS) B lymphoblastoid line in 26recombinant clones taken at random. Only MoMuLV type contaminantsequences, originating from the commercial reverse transcriptase usedfor the CDNA synthesis step, and sequences without any particularretroviral analogy were to be found in this control, as a result of the“consensus” amplification of homologous polymerase sequences which isproduced by this PCR technique. Furthermore, the absence of aconcentrated target which competes for the amplification reaction in thecontrol sample permits the amplification of dilute contaminants. Thedifference in results is manifestly highly significant (chi- squared,p<0.001).

EXAMPLE 3

[0151] Obtaining a Clone PSJ17, Defining a Retrovirus MSRV-1, byReaction of Endogenous Reverse Transcriptase with a Virion PreparationOriginating from the PLI-2 Line.

[0152] This approach is directed towards obtaining reverse-transcribedDNA sequences from the supposedly retroviral RNA in the isolate usingthe reverse transcriptase activity present in this same isolate. Thisreverse transcriptase activity can theoretically function only in thepresence of a retroviral RNA linked to a primer tRNA or hybridized withshort strands of DNA already reverse-transcribed in the retroviralparticles (16). Thus, the obtaining of specific retroviral sequences ina material contaminated with cellular nucleic acids was optimizedaccording to these authors by means of the specific enzymaticamplification of the portions of viral RNAs with a viral reversetranscriptase activity. To this end, the authors determined theparticular physicochemical conditions under which this enzymaticactivity of reverse transcription on RNAs contained in virions could beeffective in vitro. These conditions correspond to the technicaldescription of the protocols presented below (endogenous RT reaction,purification, cloning and sequencing).

[0153] The molecular approach consisted in using a preparation ofconcentrated but unpurified virion obtained from the culturesupernatants of the PLI-2 line, prepared according to the followingmethod: the culture supernatants are collected twice weekly,precentrifuged at 10,000 rpm for 30 minutes to remove cell debris andthen frozen at −80° C. or used as they are for the following steps. Thefresh or thawed supernatants are centrifuged on a cushion of 30%glycerol-PBS at 100,000 g (or 30,000 rpm in a type 45 T LKB-HITACHIrotor) for 2 h at 4° C. After removal of the supernatant, the sedimentedpellet is taken up in a small volume of PBS and constitutes the fractionof concentrated but unpurified virion. This concentrated but unpurifiedviral sample was used to perform a so-called endogenous reversetranscription reaction, as described below.

[0154] A volume of 200 μl of virion purified according to the protocoldescribed above, and containing a reverse transcriptase activity ofapproximately 1-5 million dpm, is thawed at 37° C. until a liquid phaseappears, and then placed on ice. A 5-fold concentrated buffer wasprepared with the following components: 500 mM Tris-HCl pH 8.2; 75 mMNaCl; 25 mM MgC12; 75 mM DTT and 0.10% NP 40; 100 μl of 5× buffer+25 μlof a 100 mM solution of dATP +25 ml of a 100 mM solution of dTTP+25 mlof a 100 μM solution of dGTP+25 μl of a 100 mM solution of dCTP +100 mlof sterile distilled water+200 ml of the virion suspension (RT activityof 5 million DPM) in PBS were mixed and incubated at 42° C. for 3 hours.After this incubation, the reaction mixture is added directly to abuffered phenol/- chloroform/isoamyl alcohol mixture (Sigma ref. P3803); the aqueous phase is collected and one volume of steriledistilled water is added to the organic phase to re-extract the residualnucleic acid material: The collected aqueous phases are combined, andthe nucleic acids contained are precipitated by adding 3M sodium acetatepH 5.2 to {fraction (1/10)}volume+2 volumes of ethanol+1 μl of glycogen(Boehringer-Mannheim ref. 901 393) and placing the sample at −20° C. for4 h or overnight at +4° C. The precipitate obtained after centrifugationis then washed with 70% ethanol and resuspended in 60 ml of distilledwater. The products of this reaction were then purified, cloned andsequenced according to the protocol which will now be described:blunt-ended DNAs with unpaired adenines at the ends were generated: a“filling-in” reaction was first performed: 25 μl of the previouslypurified DNA solution were mixed with 2 μl of a 2.5 mM solutioncontaining, in equimolar amounts, DATP+dGTP+dTTP+dCTP/1 μl of T4 DNApolymerase (Boehringer-Mannheim ref. 1004 786) / 5 μl of 10× “incubationbuffer for restriction enzyme” (Boehringer-Mannheim ref. 1417 975) / 1μl of a 1% bovine serum albumin solution / 16 μl of sterile distilledwater. This mixture was incubated for 20 minutes at 11° C. 50 μl of TEbuffer and 1 μl of glycogen (Boehringer-Mannheim ref. 901 393) wereadded thereto before extraction of the nucleic acids withphenol/chloroform/isoamyl alcohol (Sigma ref. P 3803) and precipitationwith sodium acetate as described above. The DNA precipitated aftercentrifugation is resuspended in 10 μl of 10 mM Tris buffer pH 7.5. 5 μlof this suspension were then mixed with 20 μl of 5× Taq buffer, 20 μl of5 mM dATP, 1 μl (5U) of Taq DNA polymerase (Amplitaq™) and 54 μl ofsterile distilled water. This mixture is incubated for 2 h at 75° C.with a film of oil on the surface of the solution. The DNA suspended inthe aqueous solution drawn off under the film of oil after incubation isprecipitated as described above and resuspended in 2 μl of steriledistilled water. The DNA obtained was inserted into a plasmid using theTA Cloning™ kit. The 2 μl of DNA solution were mixed with 5 μl ofsterile distilled water, 1 μl of a 10-fold concentrated ligation buffer“10× LIGATION BUFFER”, 2 μl of “pCR™ VECTOR” (25 ng/ml) and 1 μl of “TADNA LIGASE”. This mixture was incubated overnight at 12° C. Thefollowing steps were carried out according to the instructions of the TACloning® kit (British Biotechnology). At the end of the procedure, thewhite colonies of recombinant bacteria (white) were picked out in orderto be cultured and to permit extraction of the plasmids incorporatedaccording to the so-called “miniprep” procedure (17). The plasmidpreparation from each recombinant colony was cut with a suitablerestriction enzyme and analysed on agarose gel. Plasmids possessing aninsert detected under UV light after staining the gel with ethidiumbromide were selected for sequencing of the insert, after hybridizationwith a primer complementary to the Sp6 promoter present on the cloningplasmid of the TA cloning kit®. The reaction prior to sequencing wasthen performed according to the method recommended for the use of thesequencing kit “Prism ready reaction kit dye deoxyterminator cyclesequencing kit” (Applied Biosystems, ref. 401384), and automaticsequencing was carried out with an Applied Biosystems “AutomaticSequencer, model 373 A” apparatus according to the manufacturer'sinstructions.

[0155] Discriminating analysis on the computerized data banks of thesequences cloned from the DNA fragments present in the reaction mixtureenabled a retroviral type sequence to be revealed. The correspondingclone PSJ17 was completely sequenced, and the sequence obtained,presented in FIG. 6 and identified by SEQ ID No. 9, was analysed usingthe “Geneworks®” software on the updated “Genebank®” data banks. Anidentical sequence already described could not be found by analysis ofthe data banks. Only a partial homology with some known retroviralelements was to be found. The most useful relative homology relates toan endogenous retrovirus designated ERV-9, or HSERV-9, according to thereferences (18).

EXAMPLE 4

[0156] PCR Amplification of the Nucleic Acid Sequence Contained Betweenthe 5′ Region Defined by the Clone *POL NSRV-1B′ and the 3′ REGIONDEFINED BY THE CLONE PSJ17.

[0157] Five oligonucleotides, M001, M002-A, M003-BCD, P004 and P005,were defined in order to amplify the RNA originating from purified POL-2virions. Control reactions were performed so as to check for thepresence of contaminants (reaction with water). The amplificationconsists of an RT-PCR step according to the protocol described inExample 2, followed by a “nested” PCR according to the PCR protocoldescribed in the document EP-A-0,569,272. In the first RT-PCR cycle, theprimers M001 and P004 or P005 are used. In the second PCR cycle, theprimers M002-A or M003-BCD and the primer P004 are used. The primers arepositioned as follows:              M002-A              M003-BCD       M001               P004  P005                                                                             RNA POL-2<-------------->       <----------------------->      polMSRV-1B            PSJ17 primer GGTCITICCICAIGG (SEQ ID NO:20) M001:primer TTAGGGATAGCCCTCATCTCT (SEQ ID NO:21) M002-A: primerTCAGGGATAGCCCCCATCTAT (SEQ ID NO:22) M003-BCD: primerAACCCTTTGCCACTACATCAATTT (SEQ ID NO:23) P004: primerGCGTAAGGACTCCTAGAGCTATT (SEQ ID NO:24) P005:

[0158] The “nested” amplification product obtained, and designatedM003-P004, is presented in FIG. 7, and corresponds to the sequence SEQID NO:8.

EXAMPLE 5

[0159] Amplification and Cloning of a Portion of the MSRV-1 RetroviralGenome using a Sequence Already Identified, in a Sample of VirusPurified at the Peak of Reverse Transcriptinve Activity

[0160] A PCR technique derived from the technique published by Frohman(19) was used. The technique derived makes it possible, using a specificprimer at the 3′ end of the genome to be amplified, to elongate thesequence towards the 5′ region of the genome to be analysed. Thistechnical variant is described in the documentation of the firm“Clontech Laboratories Inc.”, (Palo-Alto California, USA) supplied withits product “5′-AmpliFINDER™ RACE Kit”, which was used on a fraction ofvirion purified as described above.

[0161] The specific 3′ primers used in the kit protocol for thesynthesis of the cDNA and the PCR amplification are, respectively,complementary to the following MSRV-1 sequences: cDNA:TCATCCATGTACCGAAGG (SEQ ID NO:25) amplification: ATGGGGTTCCCAAGTTCCCT(SEQ ID NO:26)

[0162] The products originating from the PCR were purified afterpurification on agarose gel according to conventional methods (17), andthen resuspended in 1.0 ml of distilled water. Since one of theproperties of Taq polymerase consists in adding an adenine at the 3′ endof each of the two DNA strands, the DNA obtained was inserted directlyinto a plasmid using the TA Cloning™ kit (British Biotechnology). The 2μl of DNA solution were mixed with 5 μl of sterile distilled water, 1 μlof a 10-fold concentrated ligation buffer “10′ LIGATION BUFFER”, 2 μl of“pCR™ VECTOR” (25 ng/ml) and 1 μl of “TA DNA LIGASE”. This mixture wasincubated overnight at 12° C. The following steps were carried outaccording to the instructions of the TA Cloning® kit (BritishBiotechnology). At the end of the procedure, the white colonies ofrecombinant bacteria (white) were picked out in order to be cultured andto permit extraction of the plasmids incorporated according to theso-called “miniprep” procedure (17). The plasmid preparation from eachrecombinant colony was cut with a suitable restriction enzyme andanalysed on agarose gel. Plasmids possessing an insert detecte under UVlight after staining the gel with ethidium bromide were selected forsequencing of the insert, after hybridization with a primercomplementary to the Sp6 promoter present on the cloning plasmid of theTA Cloning Kit®. The reaction prior to sequencing was then performedaccording to the method recommended for the use of the sequencing kit“Prism ready reaction kit dye deoxyterminator cycle sequencing kit”(Applied Biosystems, ref. 401384), and automatic sequencing was carriedout with an Applied Biosystems “Automatic Sequencer model 373 A”apparatus according to the manufacturer's instructions.

[0163] This technique was applied first to two fractions of virionpurified as described below on sucrose from the “POL-2” isolate producedby the PLI-2 line on the one hand, and from the MS7PG isolate producedby the LM7PC line on the other hand. The culture supernatants arecollected twice weekly, precentrifuged at 10,000 rpm for 30 minutes toremove cell debris and then frozen at −80° C. or used as they are forthe following steps. The fresh or thawed supernatants are centrifuged ona cushion of 30% glycerol-PBS at 100,000 g (or 30,000 rpm in a type 45 TLKB-HITACHI rotor) for 2 h at 4° C. After removal of the supernatant,the sedimented pellet is taken up in a small volume of PBS andconstitutes the fraction of concentrated but unpurified virions. Theconcentrated virus is then applied to a sucrose gradient in sterile PBSbuffer (15 to 50% weight/weight) and ultracentrifuged at 35,000 rpm(100,000 g) for 12 h at +4° C., in a swing-out rotor. 10 fractions arecollected, and 20 μl are withdrawn from each fraction afterhomogenization to assay the reverse transcriptase activity thereinaccording to the technique described by H. Perron (3). The fractionscontaining the peak of “LM7-like” RT activity are then diluted insterile PBS buffer and ultracentrifuged for one hour at 35,000 rpm(100,000 g) to sediment the viral particles. The pellet of purifiedvirion thereby obtained is then taken up in a small volume of a bufferwhich is appropriate for the extraction of RNA. The cDNA synthesisreaction mentioned above is carried out on this RNA extracted frompurified extracellular virion. PCR amplification according to thetechnique mentioned above enabled the clone F1-11 to be obtained, whosesequence, identified by SEQ ID NO:2, is presented in FIG. 8.

[0164] This clone makes it possible to define, with the different clonespreviously sequenced, a region of considerable length (1.2 kb)representative of the “pol” gene of the MSRV-1 retrovirus, as presentedin FIG. 9. This sequence, designated SEQ ID NO:1, is reconstituted fromdifferent clones overlapping one another at their ends, correcting theartefacts associated with the primers and with the amplification orcloning techniques which would artificially interrupt the reading frameof the whole. This sequence will be identified below under thedesignation “MSRV-1 pol* region”. Its degree of homology with theHSERV-9 sequence is shown in FIG. 12.

[0165] In FIG. 9, the potential reading frame with its translation intoamino acids is presented below the nucleic acid sequence.

EXAMPLE 6

[0166] Detection of Specific MSRV-1 and MSRV-2 Sequences in DifferentSamlpes of Plasma Originating from Patients Suffering from MS or fromControls.

[0167] A PCR technique was used to detect the MSRV-1 and MSRV-2 genomesin plasmas obtained after taking blood samples from patients sufferingfrom MS and from non-MS controls onto EDTA.

[0168] Extraction of the RNAs from plasma was performed according to thetechnique described by P. Chomzynski (20), after adding one volume ofbuffer containing guanidinium thiocyanate to 1 ml of plasma storedfrozen at −80° C. after collection.

[0169] For MSRV-2, the PCR was performed under the same conditions andwith the following primers: 5′ primer, identified by5′ GTAGTTCGATGTAGAAAGCG 3′; SEQ ID NO:14 3′ primer, identified by5′ GCATCCGGCAACTGCACG 3′. SEQ ID NO:15

[0170] However, similar results were also obtained with the followingPCR primers in two successive amplifica tions by “nested” PCR on samplesof nucleic acids not treated with DNase.

[0171] The primers used for this first step of 40 cycles with ahybridization temperature of 48° C. are the following:

[0172] 5′ primer, identified by SEQ ID NO:27 5′ GCCGATATCACCCGCCATGG 3′,corresponding to a 5′ MSRV-2 PCR primer, for a first PCR on samples frompatients,

[0173] 3′ primer, identified by SEQ ID NO:28 5′ GCATCCGGCAACTGCACG 3′,corresponding to a 3′ MSRV-2 PCR primer, for a first PCR on samples frompatients.

[0174] After this step, 10 μl of the amplification product are taken andused to carry out a second, so-called “nested” PCR amplification withprimers located within the region already amplified. This second steptakes place over 35 cycles, with a primer hybridization (“annealing”)temperature of 50° C. The reaction volume is 100 μl.

[0175] The primers used for this second step are the 10 following:

[0176] 5′ primer, identified by SEQ ID NO:29 5′ CGCGATGCTGGTTGGAGAGC 3′,corresponding to a 5′ MSRV-2 PCR primer, for a nested PCR on samplesfrom patients,

[0177] 3′ primer, identified by SEQ ID NO:30 5′TCTCCACTCCGAATATTCCG 3′,corresponding to a 3′ MSRV-2 PCR primer, for a nested PCR on samplesfrom patients.

[0178] For MSRV-1, the amplification was performed in two steps.Furthermore, the nucleic acid sample is treated beforehand with DNase,and a control PCR without RT (AMV reverse transcriptase) is performed onthe two amplification steps so as to verify that the RT-PCRamplification comes exclusively from the MSRV-1 RNA. In the event of apositive control without RT, the initial aliquot sample of RNA is againtreated with DNase and amplified again.

[0179] The protocol for treatment with DNase lacking RNAse activity isas follows: the extracted RNA is aliquoted in the presence of “RNAseinhibitor” (Boehringer-Mannheim) in water treated with DEPC at a finalconcentration of 1 μg in 10 μl; to these 10 μl, 1 μl of “RNAse-freeDNAse” (Boehringer-Mannheim) and 1.2 μl of pH 5 buffer containing 0.1M/1 sodium acetate 35 and 5 mM/l MgSO₄ is added; the mixture isincubated for 15 min at 20° C. and brought to 95° C. for 1.5 min in a“thermocycler”.

[0180] The first MSRV-1 RT-PCR step is performed according to a variantof the RNA amplification method as described in Patent Application No.EP-A-0,569,272. In particular, the CDNA synthesis step is performed at42° C. for one hour; the PCR amplification takes place over 40 cycles,with a primer hybridization (“annealing”) temperature of 53° C. Thereaction volume is 100 μl.

[0181] The primers used for this first step are the following:5′ primer, identified by 5′ AGGAGTAAGGAAACCCAACGGAC 3′; SEQ ID NO:163′ primer, identified by 5′ TAAGAGTTGCACAAGTGCG 3′. SEQ ID NO:17

[0182] After this step, 10 μl of the amplification product are taken andused to carry out a second, so-called “nested” PCR amplification withprimers located within the region already amplified. This second steptakes place over 35 cycles, with a primer hybridization (“annealing”)temperature of 53° C. The reaction volume is 100 μl.

[0183] The primers used for this second step are the following:5′ primer, identified by 5′ TCAGGGATAGCCCCCATCTAT 3′; SEQ ID NO:183′ primer, identified by 5′ AACCCTTTGCCACTACATCAATTT 3′. SEQ ID NO:19

[0184]FIGS. 10 and 11 present the results of PCR in the form ofphotographs under ultraviolet light of ethidium bromide-impregnatedagarose gels, in which an electrophoresis of the PCR amplificationproducts applied separately to the different wells was performed.

[0185] The top photograph (FIG. 10) shows the result of specific MSRV-2amplification.

[0186] Well number 8 contains a mixture of DNA molecular weight markers,and wells 1 to 7 represent, in order, the products amplified from thetotal RNAs of plasmas originating from 4 healthy controls free from MS(wells 1 to 4) and from 3 patients suffering from MS at different stagesof the disease (wells 5 to 7).

[0187] In this series, MSRV-2 nucleic acid material is detected in theplasma of one case of MS out of the 3 tested, and in none of the 4control plasmas. Other results obtained on more extensive series confirmthese results.

[0188] The bottom photograph (FIG. 11) shows the result of specificamplification by MSRV-1 “nested” RT-PCR:

[0189] well No. 1 contains the PCR product produced with water alone,without the addition of AMV reverse transcriptase; well No. 2 containsthe PCR product produced with water alone, with the addition of AMVreverse transcriptase; well number 3 contains a mixture of DNA molecularweight markers; wells 4 to 13 contain, in order, the products amplifiedfrom the total RNAs extracted from sucrose gradient fractions (collectedin a downward direction), on which gradient a pellet of virionoriginating from a supernatant of a culture infected with MSRV-1 andMSRV-2 was centrifuged to equilibrium according to the protocoldescribed by H. Perron (13); to well 14 nothing was applied; to wells 15to 17, the amplified products of RNA extracted from plasmas originatingfrom 3 different patients suffering from MS at different stages of thedisease were applied.

[0190] The MSRV-1 retroviral genome is indeed to be found in the sucrosegradient fraction containing the peak of reverse transcriptase activitymeasured according to the technique described by H. Perron (3), with avery strong intensity (fraction 5 of the gradient, placed in well No.8). A slight amplification has taken place in the first fraction (wellNo. 4), probably corresponding to RNA released by lysed particles whichfloated at the surface of the gradient; similarly, aggregated debris hassedimented in the last fraction (tube bottom), carrying with it a fewcopies of the MSRV-1 genome which have given rise to an amplification oflow intensity.

[0191] Of the 3 MS plasmas tested in this series, MSRV-1 RNA turned upin one case, producing a very intense amplification (well No. 17).

[0192] In this series, the MSRV-1 retroviral RNA genome, probablycorresponding to particles of extracellular virus present in the plasmain extremely small numbers, was detected by “nested” RT-PCR in one caseof MS out of the 3 tested. Other results obtained on more extensiveseries confirm these results.

[0193] Furthermore, the specificity of the sequences amplified by thesePCR techniques may be verified and evaluated by the “ELOSA” technique asdescribed by F. Mallet (21) and in the document FR- A-2,663,040.

[0194] For MSRV-1, the products of the nested PCR described above may betested in two ELOSA systems enabling a consensus A and a consensus B+C+Dof MSRV-1 to be detected separately, corresponding to the subfamiliesdescribed.in Example 1 and FIGS. 1 and 2. In effect, the sequencesclosely resembling the consensus B+C+D are to be found essentially inthe RNA samples originating from MSRV-1 virions purified from culturesor amplified in extracellular biological fluids of MS patients, whereasthe sequences closely resembling the consensus A are essentially to befound in normal human cellular DNA.

[0195] The ELOSA/MSRV-1 system for the capture and specifichybridization of the PCR products of the subfamily A uses a captureoligonucleotide cpV1A with an amine bond at the 5′ end and abiotinylated detection oligonucleotide dpV1A having as their sequence,respectively:

[0196] cpV1A identified by SEQ ID NO:31

[0197]5′ GATCTAGGCCACTTCTCAGGTCCAGS 3′, corresponding to the ELOSAcapture oligonucleotide for the products of MSRV-1 nested PCR performedwith the primers identified by SEQ ID NO:16 and SEQ ID NO:17, optionallyfollowed by amplification with the primers identified by SEQ ID NO18 andSEQ ID NO:19 on samples from patients;

[0198] dpV1A identified by SEQ ID NO:32;

[0199] 5′ CATCTITTTGGICAGGCAITAGC 3′, corresponding to the ELOSA captureoligonucleotide for the subfamily A of the products of MSRV-1 “nested”PCR performed with the primers identified by SEQ ID NO:16 and SEQ IDNO:17, optionally followed by amplification with the primers identifiedby SEQ ID NO:18 and SEQ ID NO:19 on samples from patients.

[0200] The ELOSA/MSRV-1 system for the capture and specifichybridization of the PCR products of the subfamily B+C+D uses the samebiotinylated detection oligonucleotide dpV1A and a captureoligonucleotide cpV1B with an amine bond at the 5′ end having as itssequence:

[0201] dpV1B identified by SEQ ID NO:33

[0202]5° CTTGAGCCAGTTCTCATACCTGGA 3′, corresponding to the ELOSA captureoligonucleotide for the subfamily B+C+D of the products of MSRV-1“nested” PCR performed with the primers identified by SEQ ID NO:16 andSEQ ID NO:17, optionally. followed by amplification with the primersidentified by SEQ ID NO:18 and SEQ ID NO:19 on samples from patients.

[0203] This ELOSA detection system enabled it to be verified that noneof the PCR products thus amplified from DNase-treated plasmas of MSpatients contained a sequence of the subfamily A, and that all werepositive with the consensus of the subfamilies B, C and D.

[0204] For MSRV-2, a similar ELOSA technique was evaluated on isolatesoriginating from infected cell cultures, using the following PCRamplification primers,

[0205] 5′ primer, identified by SEQ ID NO:34

[0206] 5′ AGTGYTRCCMCARGGCGCTGAA 3′, corresponding to a 5′ MSRV-2 PCRprimer, for PCR on samples from cultures,

[0207] 3′ primer, identified by SEQ ID NO:35

[0208] 5′ GMGGCCAGCAGSAKGTCATCCA 3′, corresponding to a 3′ MSRV-2 PCRprimer, for PCR on samples from cultures,

[0209] and the capture oligonucleotides with an amine bond at the 5′ endcpV2.and the biotinylated detection oligonucleotide dpV2 having as theirrespective sequences:

[0210] cpV2 identified by SEQ ID NO:36

[0211] 5 GGATGCCGCCTATAGCCTCTAC 3′, corresponding to an ELOSA captureoligonucleotide for the products of MSRV-2 PCR performed with theprimers SEQ ID NO:34 and SEQ ID NO:35, or optionally with the degenerateprimers defined by Shih (12).

[0212] dpV2 identified by SEQ ID NO:37

[0213] 5′ AAGCCTATCGCGTGCAGTTGCC 3′, corresponding to an ELOSA detectionoligonucleotide for the products of MSRV-2 PCR performed with theprimers SEQ ID NO:34 and SEQ ID NO:35, or optionally with the degenerateprimers defined by Shih (12)

[0214] This PCR amplification system with a pair of primers differentfrom those which were described previously for amplification on thesamples from patients made it possible to confirm the infection withMSRV-2 of in vitro cultures and of samples of nucleic acids used for themolecular biology studies.

[0215] All things considered, the first results of PCR detection of thegenome of pathogenic and/or infective agents show that it is possiblethat free “virus” may circulate in the blood stream of patients in anacute, virulent phase, outside the nervous system. This is compatiblewith the almost invariable presence of “gaps” in the blood-brain barrierof patients in an active phase of MS.

EXAMPLE 7

[0216] Obtaining Sequences of the “env” Gene of the MSRV-1 RetroviralGenome

[0217] As has already been described in Example 5, a PCR techniquederived from the technique published by Frohman (19) was used. Thetechnique derived makes it possible, using a specific primer at the 3′end of the genome to be amplified, to elongate the sequence towards the5′ region of the genome to be analysed. This technical variant isdescribed in the documentation of “Clontech Laboratories Inc.,(Palo-Alto Calif., USA) supplied with its product “5′-AmpliFINDER™ RACEKit”, which was used on a fraction of virion purified as describedabove.

[0218] In order to carry out an amplification of the 3′ region of theMSRV-1 retroviral genome encompassing the region of the “env” gene, astudy was carried out to determine a consensus sequence in the LTRregions of the same type as those of the defective endogenous retrovirusHSERV-9 (18, 24), with which the MSRV-1 retrovirus displays partialhomologies.

[0219] The same specific 3′ primer was used in the kit protocol for thesynthesis of the CDNA and the PCR amplification; its sequence is asfollows:

[0220] GTGCTGATTGGTGTATTTACAATCC (SEQ ID NO 45)

[0221] Synthesis of the complementary DNA (CDNA) and unidirectional PCRamplification with the above primer were carried out in one stepaccording to the method described in Patent EP-A-0,569,272.

[0222] The products originating from the PCR were extracted afterpurification of agarose gel according to conventional methods (17), andthen resuspended in 10 ml of distilled water. Since one of theproperties of Taq polymerase consists in adding an adenine at the 3′ endof each of the two DNA strands, the DNA obtained was inserted directlyinto a plasmid using the TA Cloning™ kit (British Biotechnology). The 2μl of DNA solution were mixed with 5 μl of sterile distilled water, 1 μlof a 10-fold concentrated ligation buffer “10× LIGATION BUFFER”, 2 μl of“pCR™ VECTOR” (25 ng/ml) and 1 μl of “TA DNA LIGASE”. This mixture wasincubated overnight at 12° C. The following steps were carried outaccording to the instructions of the TA Cloning® kit (BritishBiotechnology). At the end of the procedure, the white colonies ofrecombinant bacteria (white) were picked out in order to be cultured andto permit extraction of the plasmids incorporated according to theso-called “miniprep” procedure (17). The plasmid preparation from eachrecombinant colony was cut with a suitable restriction enzyme andanalysed on agarose gel. Plasmids possessing an insert detected under UVlight after staining the gel with ethidium bromide were selected forsequencing of the insert, after hybridization with a primercomplementary to the Sp6 promoter present on the cloning plasmid of theTA Cloning Kit®. The reaction prior to sequencing was then performedaccording to the method recommended for the use of the sequencing kit“Prism ready reaction kit dye deoxyterminator cycle sequencing kit”(Applied Biosystems, ref. 401384), and automatic sequencing was carriedout with an Applied Biosystems “automatic sequencer, model 373 A[lacuna] apparatus according to the manufacturer's instructions.

[0223] This technical approach was applied to a sample of virionconcentrated as described below from a mixture of culture supernatantsproduced by B lymphoblastoid lines such as are described in Example 2,established from lymphocytes of patients suffering from MS andpossessing reverse transcriptase activity which is detectable accordingto the technique described by Perron et al. (3): the culturesupernatants are collected twice weekly, precentrifuged at 10,000 rpmfor 30 minutes to remove cell debris and then frozen at −80° C. or usedas they are for the following steps. The fresh or thawed supernatantsare centrifuged on a cushion of 30% glycerol-PBS at 100,000 g for 2 h at4° C. After removal of the supernatant, the sedimented pelletconstitutes the sample of concentrated but unpurified virions. Thepellet thereby obtained is then taken up in a small volume of anappropriate buffer for the extraction of RNA. The cDNA synthesisreaction mentioned above is carried out on this RNA extracted fromconcentrated extracellular virion.

[0224] RT-PCR amplification according to the technique mentioned aboveenabled the clone FBd3 to be obtained, whose sequence, identified by SEQID NO:46, is presented in FIG. 13.

[0225] In FIG. 14, the sequence homology between the clone FBd3 and theHSERV-9 retrovirus is shown on the matrix chart by a continuous line forany partial homology greater than or equal to 65%. It can be seen thatthere are homologies in the flanking regions of the clone (with the polgene at the 5′ end and with the env gene and then the LTR at the 3′end), but that the internal region is totally divergent and does notdisplay any homology, even weak, with the “env” gene of HSERV9.Furthermore, it is apparent that the clone FBd3 contains a longer “env”region than the one which is described for the defective endogenousHSERV-9; it may thus be seen that the internal divergent regionconstitutes an “insert” between the regions of partial homology with theHSERV-9 defective genes.

EXAMPLE 8

[0226] Amplification, Cloning and Sequencing of the Region of the MSRV-1Retroviral Genome Located Between the Clones PSJ17 and FBd3

[0227] Four oligonucleotides, F1, B4, F6 and B1, were defined foramplifying RNA originating from concentrated virions of the strains POL2and MS7PG. Control reactions were performed so as to check for thepresence of contaminants (reaction with water). The amplificationconsists of a first step of RT-PCR according to the protocol describedin Patent Application EP-A-0,569,272, followed by a second step of PCRperformed on 10 ml of product of the first step with primers internal tothe amplified first region (“nested” PCR). In the first RT-PCR cycle,the primers F1 and B4 are used. In the second PCR cycle, the primers F6and the primer B1 are used. The primers are positioned as follows:     F1   F6                      B1   B4                                                                                     RNA MSRV-1PSJ17                           FBd3---------->           <------------------/--- 5′polMSRV-1          3′pol MSRV-1      / 5′env Their composition is: primerTGATGTGAACGGCATACTCACTG (SEQ ID NO:47) F1: primerCCCAGAGGTTAGGAACTCCCTTTC (SEQ ID N0 48) B4: primerGCTAAAGGAGACTTGTGGTTGTCAG (SEQ ID N0 49) F6: primerCAACATGGGCATTTCGGATTAG (SEQ ID N0 50) B1:

[0228] The product of “nested” amplification obtained and designated “tpol” is presented in FIG. 15, and corresponds to the sequence SEQ IDNO:51.

EXAMPLE 9

[0229] Obtaining New Sequences, Expressed as RNA in Cells in CultureProducing MSRV-1, and Comprising an “env” Region of the NSRV-1Retroviral Genome

[0230] A library of cDNA was produced according to the proceduredescribed by the manufacturer of the “cDNA synthesis module, cDNA rapidadaptator ligation module, CDNA rapid cloning module and lambda gt10 invitro packaging module” kits (Amersham, ref RPN1256Y/Z, RPN1712,RPN1713, RPN1717, N334Z), from the messenger RNA extracted from cells ofa B lymphoblastoid line such as is described in Example 2, establishedfrom the lymphocytes of a patient suffering from MS and possessingreverse transcriptase activity which is detectable according to thetechnique described by Perron et al. (3).

[0231] Oligonucleotides were defined for amplifying the cDNA cloned intothe nucleic acid library between the 3′ region of the clone PSJ17 (pol)and the 5′ (LTR) region of the clone FBd3. Control reactions wereperformed so as to check for the presence of contaminants (reaction withwater). PCR reactions performed on the nucleic acids cloned into thelibrary with different pairs of primers enabled a series [lacuna] cloneslinking pol sequences to the MSRV-1 type env or LTR sequences to beamplified.

[0232] Two clones are representative of the sequences obtained in thecellular cDNA library:

[0233] the clone JLBc1, whose sequence SEQ ID NO:52 is presented in FIG.16;

[0234] the clone JLBc2, whose sequence SEQ ID NO:53 is presented in FIG.17.

[0235] The sequences of the clones JLBc1 and JLBc2 are homologous tothat of the clone FBd3, as is apparent in FIGS. 18 and 19. The homologybetween the clone JLBc1 and the clone JLBc2 is shown in FIG. 20.

[0236] The homologies between the clones JLBc1 and JLBc2 on the one handand the HSERV9 sequence on the other hand are presented, respectively,in FIGS. 21 and 22.

[0237] It will be noted that the region of homology between JLB1, JLB2and FBd3 comprises, with a few sequence and size variations of the“insert”, the additional sequence absent (“inserted”) in the HSERV-9 envsequence, as described in Example. 8.

[0238] It will also be noted that the cloned “pol” region is veryhomologous to HSERV-9, does not possess a reading frame (bearing in mindthe sequence errors induced by the techniques used, including even theautomatic sequencer) and diverges from the MSRV-1 sequences obtainedfrom virions. In view of the fact that these sequences were cloned fromthe RNA of cells expressing MSRV-1 particles, it is probable that theyoriginate from endogenous retroviral elements related to the ERV9family; this is all the more likely for the fact that the pol and envgenes are present on the same RNA which is clearly not the MSRV-1genomic RNA. Some of these ERV9 elements possess functional LTRs whichcan be activated by replicative viruses coding for homologous orheterologous transactivators. Under these conditions, the relationshipbetween MSRV-1 and HSERV-9 makes probable the transactivation of thedefective (or otherwise) endogenous ERV9 elements by homologous, or evenidentical, MSRV-1 transactivating proteins.

[0239] Such a phenomenon may induce a viral interference between theexpression of MSRV-1 and the related endogenous elements. Such aninterference generally leads to a so-called “defective-interfering”expression, some features of which were to be found in theMSRV-1-infected cultures studied. Furthermore, such a phenomenon doesnot lack generation of the expression of polypeptides, or even ofendogenous retroviral proteins which are not necessarily tolerated bythe immune system. Such a scheme of aberrant expression of endogenouselements related to MSRV-1 and induced by the latter is liable tomultiply the aberrant antigens, and hence to contribute to the inductionof autoimmune processes such as are observed in MS.

[0240] It is, however, essential to note that the clones JLBc1 and JLBc2differ from the ERV9 or HSERV9 sequence already described, in that theypossess a longer env region comprising an additional region totallydivergent from ERV9. Their kinship with the endogenous ERV9 family mayhence be defined, but they clearly constitute novel elements neverhitherto described. In effect, interrogation of the data banks ofnucleic acid sequences available in version No. 15 (1995) of the“Entrez” software (NCBI, NIH, Bethesda, USA) did not enable a knownhomologous sequence in the env region of these clones to be identified.

EXAMPLE 10

[0241] OBTAINING SEQUENCES LOCATED IN TEE 5′ pol and 3′ gag REGION OFTHE NSRV-1 RETROVIRAL GENOME As has already been described in Example 5,a PCR technique derived from the technique published by Frohman (19) wasused. The technique derived makes it possible, using a specific primerat the 3′ end of the genome to be amplified, to elongate the sequencetowards the 5′ region of the genome to be analysed. This technicalvariant is described in the documentation of the firm “ClontechLaboratories Inc., (Palo-Alto California, USA) supplied with its product“5′ -AmpliFINDER™ RACE Kit”, which was used on a fraction of virionpurified as described above.

[0242] In order to carry out an amplification of the 5′ region of theMSRV-1 retroviral genome starting from the pol sequence alreadysequenced (clone F11-1) and extending towards the gag gene, MSRV-1specific primers were defined.

[0243] The specific 3′ primers used in the kit protocol for thesynthesis of the CDNA and the PCR amplification are, respectively,complementary to the following MSRV-1 sequences: cDNA:CCTGAGTTCTTGCACTAACCC (SEQ ID NO:54) amplification:GTCCGTTGGGTTTCCTTACTCCT (SEQ ID NO:55)

[0244] The products originating from the PCR were extracted afterpurification on agarose gel according to conventional methods (17), andthen resuspended in 10 ml of distilled water. Since one of theproperties of Taq polymerase consists in adding an adenine at the 3′ endof each ot the two DNA strands, the DNA obtained was inserted directlyinto a plasmid using the TA Cloning™ kit (British Biotechnology). The 2μl of DNA solution were mixed with 5 μl of sterile distilled water, 1 μlof a 10-fold concentrated ligation buffer “10× LIGATION BUFFER”, 2 μl of“pCR™ VECTOR” (25 ng/ml) and 1 μl of “TA DNA LIGASE”. This mixture wasincubated overnight at 12° C. The following steps were carried outaccording to the instructions of the TA Cloning® kit (BritishBiotechnology). At the end of the procedure, the white colonies ofrecombinant bacteria (white) were picked out in order to be cultured andto permit extraction of the plasmids incorporated according to theso-called “miniprep” procedure (17). The plasmid preparation from eachrecombinant colony was cut with a suitable restriction enzyme andanalysed on agarose gel. Plasmids possessing an insert detected under UVlight after staining the gel with ethidium bromide were selected forsequencing of the insert, after hybridization with a primercomplementary to the Sp6 promoter present on the cloning plasmid of theTA Cloning Kit®. The reaction prior to sequencing was then performedaccording to the method recommended for the use of the sequencing kit“Prism ready reaction kit dye deoxyterminator cycle seq uencing kit”(Applied Biosystems, ref. 401384), and automatic sequencing was carriedout with an Applied Biosystems “automatic sequencer model 373 A [lacuna]apparatus according to the manufacturer's instructions.

[0245] This technical approach was applied to a sample of virionconcentrated as described below from a mixture of culture supernatantsproduced by B lymphoblastoid lines such as are described in Example 2,established from lymphocytes of patients suffering from MS andpossessing reverse transcriptase activity- which is detectable accordingto the technique described by Perron et al. (3): the culturesupernatants are collected twice weekly, precentrifuged at 10,000 rpmfor 30 minutes to remove cell debris and then frozen at −80° C. or usedas they are for the following steps. The fresh or thawed supernatantsare centrifuged on a cushion of 30% glycerol-PBS at 100,000 g for 2 h at4° C. After removal of the supernatant, the sedimented pelletconstitutes the sample of concentrated but unpurified virions. Thepellet thereby obtained is then taken up in a small volume of anappropriate buffer for the extraction of RNA. The CDNA synthesisreaction mentioned above is carried out on this RNA extracted fromconcentrated extracellular virion.

[0246] RT-PCR amplification according to the technique mentioned aboveenabled the clone GM3 to be obtained, whose sequence, identified by SEQID NO 56, is presented in FIG. 23.

[0247] In FIG. 24, the sequence homology between the clone GMP3 and theHSERV-9 retrovirus is shown on the matrix chart by a continuous line,for any partial homology greater than or equal to 65%.

[0248] In summary, FIG. 25 shows the localization of the differentclones studied above, relative to the known ERV9 genome. In FIG. 25,since the MSRV-1 env region is longer than the reference ERV9 env gene,the additional region is shown above the point of insertion according toa “V”, on the understanding that the inserted material displays asequence and size vari-ability between the clones shown (JLBc1, JLBc2,FBd3). And FIG. 26 shows the position of different clones studied in theMSRV-1 pol* region.

[0249] By means of the clone GM3 described above, a possible readingframe could be defined, covering the whole of the pol gene, referencedaccording to SEQ ID NO:57, shown in the successive FIGS. 27a to 27 c.

EXAMPLE 11

[0250] Detection of Anti-MSRV-1 Specific Antibodies in Human Serum.

[0251] Identification of the sequence of the pol gene of the MSRV-1retrovirus and of an open reading frame of this gene enabled the aminoacid sequence SEQ ID NO:39 of a region of the said gene, referenced SEQID NO:40, to be determined (see FIG. 28).

[0252] Different synthetic peptides corresponding to fragments of theprotein sequence of MSRV-1 reverse transcriptase encoded by the pol genewere tested for their antigenic specificity with respect to sera ofpatients suffering from MS and of healthy controls.

[0253] The peptides were synthesized chemically by solid-phase synthesisaccording to the Merrifield technique (Barany G, and Merrifielsd R. B,1980, In the Peptides, 2, 1-284, Gross E and Meienhofer J, Eds.,Academic Press, New York). The practical details are those describedbelow.

a) Peptide Synthesis

[0254] The peptides were synthesized on a phenylacet-amidomethyl(PAM)/polystyrene/divinylbenzene resin (Applied Biosystems, Inc. FosterCity, CA), using an “Applied Biosystems 430A” automatic synthesizer. Theamino acids are coupled in the form of hydroxybenzo-triazole (HOBT)esters. The amino acids used are obtained from Novabiochem(Lauflerlfingen, Switzerland) or Bachem (Bubendorf, Switzerland).

[0255] The chemical synthesis was performed using a double couplingprotocol with N-methylpyrrolidone (NMP) as solvent. The peptides werecut from the resin, as well as the side-chain protective groups,simultaneously, using hydrofluoric acid (HF) in a suitable apparatus(type I cleavage apparatus, Peptide Institute, Osaka, Japan).

[0256] For 1 g of peptidyl resin, 10 ml of HF, 1 ml of anisole and 1 mlof dimethyl sulphide 5DMS are used. The mixture is stirred for 45minutes at −2° C. The HF is then evaporated off under vacuum. Afterintensive washes with ether, the peptide is eluted from the resin with10% acetic acid and then lyophilized.

[0257] The peptides are purified by preparative high performance liquidchromatography on a VYDAC C18 type column (250×21 mm) (The SeparationGroup, Hesperia, CA, USA). Elution is carried out with an acetonitrilegradient at a flow rate of 22 ml/min. The fractions collected aremonitored by an elution under isocratic conditions on a VYDAC® C18analytical column (250×4.6 mm) at a flow rate of 1 ml/min. Fractionshaving the same retention time are pooled and lyophilized. Thepreponderant fraction is then analysed by analytical high performanceliquid chromatography with the system described above. The peptide whichis considered to be of acceptable purity manifests itself in a singlepeak representing not less than 95% of the chromatogram.

[0258] The purified peptides are then analysed with the object ofmonitoring their amino acid composition, using an Applied Biosystems420H automatic amino acid analyser. Measurement of the (average)chemical molecular mass of the peptides is obtained using LSIMS massspectrometry in the positive ion mode on a VG. ZAB.ZSEQ double focusinginstrument connected to a DEC-VAX 2000 acquisition system (VG analyticalLtd, Manchester, England).

[0259] The reactivity of the different peptides was tested against seraof patients suffering from MS and against sera of healthy controls. Thisenabled a peptide designated POL2B to be selected, whose sequence isshown in FIG. 28 in the identifier SEQ ID NO:39, below, encoded by thepol gene of MSRV-1 (nucleotides 181 to 330).

b) Antigenic Properties

[0260] The antigenic properties of the POL2B peptide were demonstratedaccording to the ELISA protocol described below.

[0261] The lyophilized POL2B peptide was dissolved in sterile distilledwater at a concentration of 1 mg/ml. This stock solution was aliquotedand kept at +4° C. for use over a fortnight, or frozen at −20° C. foruse within 2 months. An aliquot is diluted in PBS (phosphate bufferedsaline) solution so as to obtain a final peptide concentration of 1microgram/ml. 100 microlitres of this dilution are placed in each wellof microtitration plates (“high-binding” plastic, COSTAR ref: 3590). Theplates are covered with a “plate-sealer” type adhesive and keptovernight at +4° C. for the phase of adsorption of the peptide to theplastic. The adhesive is removed and the plates are washed three timeswith a volume of 300 micro-litres of a solution A (1 ×PBS, 0.05% Tween20®), then inverted over an absorbent tissue. The plates thus drainedare filled with 200 microlitres per well of a solution B (solution A+10%of goat serum), then covered with an adhesive and incubated for 45minutes to 1 hour at 37° C. The plates are then washed three times withthe solution A as described above.

[0262] The test serum samples are diluted beforehand to {fraction(1/50)} in the solution B, and 100 microlitres of each dilute test serumare placed in the wells of each microtitration plate. A negative controlis placed in one well of each plate, in the form of 100 microlitres ofbuffer B. The plates covered with an adhesive are then incubated for 1to 3 hours at 37° C. The plates are then washed three times with thesolution A as described above. In parallel, a peroxidase-labelled goatantibody directed against human IgG (Sigma Immunochemicals ref. A6029)or IgM (Cappel ref. 55228) is diluted in the solution B (dilution{fraction (1/5000)}for the anti-IgG and {fraction (1/1000)}for theanti-IgM). 100 microlitres of the appropriate dilution of the labelledantibody are then placed in each well of the microtitration plates, andthe plates covered with an adhesive are incubated for 1 to 2 hours at37° C. A further washing of the plates is then performed as describedabove. In parallel, the peroxidase substrate is prepared according tothe directions of the “Sigma fast OPD kit” (Sigma Immunochemicals, ref.P9187). 100 microlitres of substrate solution are placed in each well,and the plates are placed protected from light for 20 to 30 minutes atroom temperature.

[0263] When the colour reaction has stabilized, the plates are placedimmediately in an ELISA plate spectrophotometric reader, and the opticaldensity (OD) of each well is read at a wavelength of 492 nm.Alternatively, 30 microlitres of 1N HCL are placed in each well to stopthe reaction, and the plates are read in the spectrophotometer within 24hours.

[0264] The serological samples are introduced in duplicate or intriplicate, and the optical density (OD) corresponding to the serumtested is calculated by taking the mean of the OD values obtained forthe same sample at the same dilution.

[0265] The net OD of each serum corresponds to the mean OD of the serumminus the mean OD of the negative control (solution B: PBS, 0.05% Tween20®, 10% goat serum).

c) Detection of Anti-MSRV-1 IgG Antibodies By ELISA:

[0266] The technique described above was used with the POLB2 peptide totest for the presence of anti-MSRV-1 specific IgG antibodies in theserum of 29 patients for whom a definite or probable diagnosis of MS wasestablished according to the criteria of Poser (23), and of 32 healthycontrols (blood donors).

[0267]FIG. 29 shows the results for each serum tested with an anti-IgGantibody. Each vertical bar represents the net optical density (OD at492 nm) of a serum tested. The ordinate axis gives the net OD at the topof the vertical bars. The first 29 vertical bars lying to the left ofthe vertical broken line represent the sera of 29 cases of MS tested,and the 32 vertical bars lying to the right of the vertical broken linerepresent the sera of 32 healthy controls (blood donors).

[0268] The mean of the net OD values for the MS sera tested is 0.62. Thediagram enables 5 controls to be revealed whose net OD rises above thegrouped values of the control population. These values may represent thepresence of specific IgGs in symptomless seropositive patients. Twomethods were hence evaluated in order to determine the statisticalthreshold of positivity of the test.

[0269] The mean of the net OD values for the controls, including thecontrols with high net OD values, is 0.36. Without the 5 controls whosenet OD values are greater than or equal to 0.5, the mean of the“negative” controls is 0.33. The standard deviation of the negativecontrols is 0.10. A theoretical threshold of positivity may becalculated according to the formula:

[0270] threshold value (mean of the net OD values of the seronegativecontrols)+( 2 or 3×standard deviation of the net OD values of theseronegative controls).

[0271] In the first case, there are considered to be symptomlessseropositives, and the threshold value is equal to 0.33+(2×0.10)=0.53.The negative results represent a non-specific “background” of thepresence of antibodies directed specifically against an epitope of thepeptide.

[0272] In the second case, if the set of controls consisting of blooddonors in apparent good health is taken as a reference basis, withoutexcluding the sera which are, on the face of it, seropositive, thestandard deviation of the “non-MS controls” is 0.116. The thresholdvalue then becomes 0.36+(2×0.116)=0.59.

[0273] According to this analysis, the test is specific for MS. In thisrespect, it is seen that the test is specific for MS, since, as shown inTable 1, no control has a net OD above this threshold. In fact, thisresult reflects the fact that the antibody titres in patients sufferingfrom MS are, for the most part, higher than in vhealthy controls whohave been in contact with MSRV-1. TABLE NO. 1 MS CONTROLS 0.681 0.35151.0425 0.56 0.5675 0.3565 0.63 0.449 0.588 0.2825 0.645 0.55 0.6635 0.520.576 0.2535 0.7765 0.55 0.5745 0.51 0.513 0.426 0.4325 0.451 0.72550.227 0.859 0.3905 0.6435 0.265 0.5795 0.4295 0.8655 0.291 0.671 0.3470.596 0.4495 0.662 0.3725 0.602 0.181 0.525 0.2725 0.53 0.426 0.5650.1915 0.517 0.222 0.607 0.395 0.3705 0.34 0.397 0.307 0.4395 0.2190.491 0.2265 0.2605 MEAN 0.62 0.33 STD DEV 0.14 0.10 THRESHOLD VALUE0.53

[0274] In accordance with the first method of calculation, and as shownin FIG. 29 and in the corresponding Table 1, 26 of the 29 MS sera give apositive result (net OD greater than or equal to 0.50), indicating thepresence of IgGs specifically directed against the POL2B peptide, henceagainst a portion of the reverse transcriptase enzyme of the MSRV-1retrovirus encoded by its pol gene, and consequently against the MSRV-1retrovirus. Thus, approximately 90% of the MS patients tested havereacted against an epitope carried by the POL2B peptide and possesscirculating IgGs directed against the latter.

[0275] Five out of 32 blood donors in apparent good health show apositive result. Thus, it is apparent that approximately 15% of thesymptomless population may have been in contact with an epitope carriedby the POL2B peptide under conditions which have led to an activeimmunization which manifests itself in the persistence of specific serumIgGs. These conditions are compatible with an immunization against theMSRV-1 retrovirus reverse transcriptase during an infection with (and/orreactivation of) the MSRV-1 retrovirus. The absence of apparentneurological pathology recalling MS in these seropositive controls mayindicate that they are healthy carriers and have eliminated aninfectious virus after immunizing themselves, or that they constitute anat-risk population of chronic carriers. In effect, epidemiological datashowing that a pathogenic agent present in the environment of regions ofhigh prevalence of MS may be the cause of this disease imply that afraction of the population free from MS has necessarily been in contactwith such a pathogenic agent. It has been shown that the MSRV-1retrovirus constitutes all or part of this “pathogenic agent” at thesource of MS, and it is hence normal for controls taken from a healthypopulation to possess IgG type antibodies against components of theMSRV-1 retrovirus. Thus, the difference in seroprevalence between the MSand control populations is extremely significant: “chi-squared”; test,p<0.001. These results hence point to an aetiopathogenic role of MSRV-1in MS.

d) Detection of Anti-MSRV-1 IgM Antibodies By ELISA

[0276] The ELISA technique with the POL2B peptide was used to test forthe presence of anti-MSRV-1 IgM specific antibodies in the serum of 36patients for whom a definite or probable diagnosis of MS was establishedaccording to the criteria of Poser (23), and of 42 healthy controls(blood donors).

[0277]FIG. 30 shows the results for each serum tested with an anti-IgMantibody. Each vertical bar represents the net optical density (OD at492 nm) of a serum tested. The ordinate axis gives the net OD at the topof the vertical bars. The first 36 vertical bars lying to the left ofthe vertical line cutting the abscissa axis represent the sera of 36cases. of MS tested, and the vertical bars lying to the right of thevertical broken line represent the sera of 42 healthy controls (blooddonors). The horizontal line drawn in the middle of the diagramrepresents a theoretical threshold defining the boundary of the positiveresults (in which the top of the bar lies above) and the negativeresults (in which the top of the bar lies below).

[0278] The mean of the net OD values for the MS cases tested is 0.19.

[0279] The mean of the net OD values for the controls is 0.09.

[0280] The standard deviation of the negative controls is 0.05.

[0281] In view of the small difference between the mean and the standarddeviation of the controls, the threshold of theoretical positivity maybe calculated according to the formula:

[0282] threshold value=(mean of the net OD values of the seronegativecontrols)+(3×standard deviation of the net OD values of the seronegativecontrols).

[0283] The threshold value is hence equal to 0.09+(3×0.05)=0.26; or, inpractice, 0.25.

[0284] The negative results represent a non-specific “background” of thepresence of antibodies directed specifically against an epitope of thepeptide.

[0285] According to this analysis, and as shown in FIG. 30 and in thecorresponding Table 2, the IgM test is specific for MS, since no controlhas a net OD above the threshold. 7 of the 36 MS sera produce a positiveIgM result; now, a study of the clinical data reveals that thesepositive sera were taken during a first attack of MS or an acute attackin untreated patients. It is known that IgMs directed against pathogenicagents are produced during primary infections or during reactivationsfollowing a latency phase of the said pathogenic agent.

[0286] The difference in seroprevalence between the MS and controlpopulations is extremely significant: “chi-squared” test, p <0.001.

[0287] These results point to an aetiopathogenic role of MSRV-1 in MS.

[0288] The detection of IgM and IgG antibodies against the POL2B peptideenables the course of an MSRV-1 infection and/or of the viralreactivation of MSRV-1 to be evaluated. TABLE NO. 2 MS CONTROLS 0.0640.243 0.087 0.11 0.044 0.098 0.115 0.028 0.089 0.094 0.025 0.038 0.0970.176 0.108 0.146 0.018 0.049 0.234 0.161 0.274 0.113 0.225 0.079 0.3140.093 0.522 0.127 0.306 0.02 0.143 0.052 0.375 0.062 0.142 0.074 0.1570.043 0.168 0.046 1.051 0.041 0.104 0.13 0.187 0.153 0.044 0.107 0.0530.178 0.153 0.114 0.07 0.078 0.033 0.118 0.104 0.177 0.187 0.026 0.0440.024 0.053 0.046 0.153 0.116 0.07 0.04 0.033 0.028 0.973 0.073 0.0080.074 0.141 0.219 0.047 0.017 MEAN 0.19 0.09 STD. DEV. 0.23 0.05THRESHOLD VALUE 0.26

e) Search for Immunodominant Epitopes in the POL2B Peptide

[0289] In order to reduce the non-specific background and to optimizethe detection of the responses of the anti-MSRV-1 antibodies, thesynthesis of octapeptides, advancing in successive one amino acid steps,covering the whole of the sequence determined by POL2B, was carried outaccording to the protocol described below.

[0290] The chemical synthesis of overlapping octapeptides covering theamino acid sequence 61-110 shown in the identifier SEQ ID NO:39 wascarried out on an activated cellulose membrane according to thetechnique of BERG et al. (1989.-J. Ann. Chem. Soc., 111, 8024-8026)marketed by Cambridge Research Biochemicals under the trade nameSpotscan. This technique permits the simultaneous synthesis of a largenumber of peptides and their analysis.

[0291] The synthesis is carried out with esterified amino acids in whichthe α-amino group is protected with an FMOC group (Nova Biochem) and theside-chain groups with protective groups such as trityl, t-butyl esteror t-butyl ether. The esterified amino acids are solubilized inN-methylpyrrolidone (NMP) at a concentration of 300 nM, and 0.9 μl areapplied to spots of deposit of bromophenol blue. After incubation for 15minutes, a further application of amino acids is carried out accordingto another 15-minute incubation. If the coupling between two amino acidshas taken place correctly, a coloration modification (change from blueto yellow-green) is observed. After three washes in DMF, an acetylationstep is performed with acetic anhydride. Next, the terminal amino groupsof the peptides in the process of synthesis are deprotected with 20%pyridine in DMF. The spots of deposit are restained with a 1% solutionof bromophenol blue in DMF, washed three times with methanol and dried.This set of operations constitutes one cycle of addition of an aminoacid, and this cycle is repeated until the synthesis is complete. Whenall the amino acids have been added, the NH2-terminal group of the lastamino acid is deprotected with 20% piperidine in DMF and acetylated withacetic anhydride. The groups protecting the side chain are removed witha dichloromethane/trifluoroacetic acid/triisobutylsilane (5 ml/5 ml/250ml) mixture. The lmmunoreactivity of the peptides is then tested byELISA.

[0292] After synthesis of the different octapeptides in duplicate on twodifferent membranes, the latter are rinsed with methanol and washed inTBS (0.1M Tris pH 7.2), then incubated overnight at room temperature ina saturation buffer. After several washes in TBS-T (0.1M Tris pH 7.2 -0.05% Tween 20), one membrane is incubated with a {fraction (1/50)}dilution of a reference serum originating from a patient suffering fromMS, and the other membrane with a {fraction (1/50)} dilution of a poolof sera of healthy controls. The membranes are incubated for 4 hours atroom temperature. After washes with TBS-T, a β-galactosidase-labelledanti-human immunoglobulin conjugate (marketed by Cambridge ResearchBiochemicals) is added at a dilution of {fraction (1/200)}, and themixture is incubated for two hours at room temperature. After washes ofthe membranes with 0.05% TBS-T and PBS, the immunoreactivity in thedifferent spots is visualized by adding 5-bromo-4-chloro-3-indolylβ-D-galactopyranoside in potassium. The intensity of coloration of thespots is estimated qualitatively with a relative value from 0 to 5 asshown in the attached FIGS. 31 to 33.

[0293] In this way, it is possible to determine two immunodominantregions at each end of the POL2B peptide, corresponding, respectively,to the amino acid sequences 65-75 (SEQ ID NO:41) and 92-109 (SEQ IDNO:42), according to FIG. 34, and lying, respectively, between theoctapeptides Phe-Cys-Ile-Pro-Val-Arg-Pro-Asp (FCIPVRPD) andArg-Pro-Asp-Ser-Gln-Phe-Leu-Phe (RPDSQFLF), andThr-Val-Leu-Pro-Gln-Gly-Phe-Arg (TVLPQGFR) andLeu-Phe-Gly-Gln-Ala-Leu-Ala-Gln (LFGQALAQ), and a region which is lessreactive but apparently more specific, since it does not produce anybackground with the control serum, represented by the octapeptidesLeu-Phe-Ala-Phe-Glu-Asp-Pro-Leu (LFAFEDPL) (SEQ ID NO:43) andPhe-Ala-Phe-Glu-Asp-Pro-Leu-Asn (FAFEDPLN) (SEQ ID NO:44).

[0294] These regions make it possible to define new peptides which aremore specific and more immunoreactive according to the usual techniques.

[0295] It is thus possible, as a result of the discoveries made and themethods developed by the inventors, to carry out a diagnosis of MSRV-1infection and/or reactivation and to evaluate a therapy in MS on thebasis of its efficacy in “negativing” the detection of these agents inthe patients' biological fluids. Furthermore, early detection inindividuals not yet displaying neurological signs of MS could make itpossible to institute a treatment which would be all the more effectivewith respect to the subsequent clinical course for the fact that itwould precede the lesion stage which corresponds to the onset ofneurological disorders. Now, at the present time, a diagnosis of MScannot be established before a symptomatology of neurological lesionshas set in, and hence no treatment is instituted before the emergence ofa clinical picture suggestive of lesions of the central nervous systemwhich are already significant. The diagnosis of an MSRV-1 and/or MSRV-2infection and/or reactivation in man is hence of decisive importance,and the present invention provides the means of doing this.

[0296] It is thus possible, apart from carrying out a diagnosis ofMSRV-1 infection and/or reactivation, to evaluate a therapy in MS on thebasis of its efficacy in “negativing” the detection of these agents inthe patients' biological fluids.

EXAMPLE 12

[0297] Obtaining a Clone LB19 Containing a Portion of the Gag Gene ofthe MSRV-1 Retrovirus

[0298] A PCR technique derived from the technique published byGonzalez-Quintial R et al. (19) and PLAZA et al. (25) was used. From thetotal RNAs extracted from a fraction of virion purified as describedabove, the cDNA was synthesized using a specific primer (SEQ ID No.64)at the 3′ end of the genome to be amplified, using EXPAND™ REVERSETRANSCRIPTASE (BOEHRINGER MANNHEIM).

[0299] cDNA: AAGGGGCATG GACGAGGTGG TGGCTTATTT (SEQ ID NO:65) (antisense)

[0300] After purification, a poly(G) tail was added at the 5′ end of theCDNA using the “Terminal transferases kit” marketed by the companyBoehringer Mannheim, according to the manufacturer's protocol.

[0301] An anchoring PCR was carried out using the following 5′ and 3′primers: AGATCTGCAG AATTCGATAT CACCCCCCCC CCCCCC (SEQ ID No. 91)(sense), and AAATGTCTGC GGCACCAATC TCCATGTT (SEQ ID No. 64) (antisense)

[0302] Next, a semi-nested anchoring PCR was carried out with thefollowing 5′ and 3′ primers: AGATCTGCAG AATTCGATAT CA (SEQ ID No.92)(sense), and AAATGTCTGC GGCACCAATC TCCATGTT (SEQ ID No.64) (antisense)

[0303] The products originating from the PCR were purified afterpurification on agarose gel according to conventional methods (17), andthen resuspended in 10 microlitres of distilled water. Since one of theproperties of Taq polymerase consists in adding an adenine at the 3′ endof each of the two DNA strands, the DNA obtained was inserted directlyinto a plasmid using the TA CloningTm kit (British Biotechnology). The 2μl of DNA solution were mixed with 5 μl of sterile distilled water, 1 μlof 10-fold concentrated ligation buffer “10×LIGATION BUFFER”, 2 μl of“pCR™ VECTOR” (25 ng/ml) and 1 μl of “T4 DNA LIGASE”. This mixture wasincubated overnight at 12° C. The following steps were carried outaccording to the instructions of the TA Cloning® kit (BritishBiotechnology). At the end of the procedure, the white colonies ofrecombinant bacteria (white) were picked out in order to be cultured andto permit extraction of the plasmids incorporated according to theso-called “miniprep” procedure (17). The plasmid preparation from eachrecombinant colony was cut with a suitable restriction enzyme andanalysed on agarose gel. Plasmids possessing an insert detected under UVlight after staining the gel with ethidium bromide were selected forsequencing of the insert, after hybridization with a primercomplementary to the Sp6 promoter present on the cloning plasmid of theTA Cloning Kit®. The reaction prior to sequencing was then performedaccording to the method recommended for the use of the sequencing kit“Prism ready reaction kit dye deoxyterminator cycle sequencing kit”(Applied Biosystems, ref. 401384), and automatic sequencing was carriedout with an Applied Biosystems “Automatic Sequencer, model 373 A”apparatus according to the manufacturer's instructions.

[0304] PCR amplification according to the technique mentioned above wasused on a cDNA synthesized from the nucleic acids of fractions ofinfective particles purified on a sucrose gradient, according to thetechnique described by H. Perron (13), from culture supernatants of Blymphocytes of a patient suffering from MS, immortalized withEpstein-Barr virus (EBV) strain B95 and expressing retroviral particlesassociated with reverse transcriptase activity as described by Perron etal. (3) and in French Patent Applications MS 10, 11 and 12. the cloneLB19, whose sequence, identified by SEQ ID NO:59, is presented in FIG.35.

[0305] The clone makes it possible to define, with the clone GM3previously sequenced and the clone G+E+A (see Example 15), a region of690 base pairs representative of a significant portion of the gag geneof the MSRV-1 retrovirus, as presented in FIG. 36. This sequencedesignated SEQ ID NO:88 is reconstituted from different clonesoverlapping at their ends. This sequence is identified under the nameMSRV-1 “gag*” region. In FIG. 36, a potential reading frame with thetranslation into amino acids is presented below the nucleic acidsequence.

EXAMPLE 13

[0306] Obtaining a Clone FBd13 Containing a pol Gene Region Related tothe MSRV-1 Retrovirus and an Apparently Incomplete ENV Region Containinga Potential Reading Frame (ORF) for a Glycoprotein.

Extraction of viral RNAs

[0307] The RNAs were extracted according to the method briefly describedbelow.

[0308] A pool of culture supernatant of B lymphocytes of patientssuffering from MS (650 ml) is centrifuged for 30 minutes at 10,000 g.The viral pellet obtained is resuspended in 300 microlitres of PBS/10 mMMgCl2. The material is treated with a DNAse (100 mg/ml)/RNAse (50 mg/ml)mixture for 30 minutes at 37° C. and then with proteinase K (50 mg/ml)for 30 minutes at 46° C.

[0309] The nucleic acids are extracted with one volume of a phenol/0.1%SDS (V/V) mixture heated to 60° C., and then re-extracted with onevolume of phenol/chloroform (1:1; V/V).

[0310] Precipitation of the material is performed with 2.5 V of ethanolin the presence of 0.1 V of sodium acetate pH =5.2. The pellet obtainedafter centrifugation is resuspended in 50 microlitres of sterile DEPCwater.

[0311] The sample is treated again with 50 mg/ml of “RNAse free” DNAsefor 30 minutes at room temperature, extracted with one volume ofphenol/chloroform and precipitated in the presence of sodium acetate andethanol.

[0312] The RNA obtained is quantified by an OD reading at 260 nm. Thepresence of MSRV-1 and the absence of DNA contaminant is monitored by aPCR and an MSRV-1-specific RTPCR associated with a specific ELOSA forthe MSRV-1 genome.

Synthesis of cDNA

[0313] 5 mg of RNA are used to synthesize a cDNA primed with a poly(DT)oligonucleotide according to the instructions of the “cDNA SynthesisModule” kit (ref RPN 1256, Amersham) with a few modifications: Thereverse transcription is performed at 45° C. instead of the recommended42° C.

[0314] The synthesis product is purified by a double extraction and adouble purification according to the manufacturer's instructions.

[0315] The presence of MSRV-1 is verified by an MSRV-1 PCR associatedwith a specific ELOSA for the MSRV-1 genome.

“Long Distance PCR”: (LD-PCR)

[0316] 500 ng of cDNA are used for the LD-PCR step (Expand Long TemplateSystem; Boehringer (ref.1681 842)).

[0317] Several pairs of oligonucleotides were used. Among these, thepair defined by the following primers: 5′ primer: GGAGAAGAGC AGCATAAGTGG (SEQ ID No. 66) 3′ primer: GTGCTGATTG GTGTATTTAC AATCC (SEQ ID No.67).

[0318] The amplification conditions are as follows:

[0319] 94° C. 10 seconds

[0320] 56° C. 30 seconds

[0321] 68° C. 5 minutes;

[0322] 10 cycles, then 20 cycles with an increment of 20 seconds in eachcycle on the elongation time. At the end of this first amplification, 2microlitres of the amplification product are subjected to a secondamplification under the same conditions as before.

[0323] The LD-PCR reactions are conducted in a Perkin model 9600 PCRapparatus in thin-walled microtubes (Boehringer).

[0324] The amplification products are monitored by electrophoresis of⅕th of the amplification volume (10 microlitres) in 1% agarose gel. Forthe pair of primers described above, a band of approximately 1.7 Kb isobtained.

Cloning of the Amplified Fragment:

[0325] The PCR product was purified by passage through a preparativeagarose gel and then through a Costar column (Spin; D. Dutcher)according to the supplier's instructions. 2 microlitres of the purifiedsolution are joined up with 50 ng of vector PCRII according to thesupplier's instructions (TA Cloning Kit; British Biotechnology)).

[0326] The recombinant vector obtained is isolated by transformation ofcompetent DH5aF′ bacteria. The bacteria are selected using theirresistance to ampicillin and the loss of metabolism for Xgal (=whitecolonies). The molecular structure of the recombinant vector isconfirmed by plasmid minipreparation and hydrolysis with the enzymeEcoR1.

[0327] FBd13, a positive clone for all these criteria, was selected. Alarge-scale preparation of the recombinant plasmid was performed usingthe Midiprep Quiagen kit (ref 12243) according to the supplier'sinstructions.

[0328] Sequencing of the clone FBd13 is performed by means of the PerkinPrism Ready Amplitaq FS dye terminator kit (ref. 402119) according tothe manufacturer's instructiions. The sequence reactions are introducedinto a Perkin type 377 or 373A automatic sequencer. The sequencingstrategy consists in gene walking carried out on both strands of theclone Fbd13.

[0329] The sequence of the clone FBd13 is identified by SEQ ID NO 58.

[0330] In FIG. 37, the sequence homology between the clone FBd13 and theHSERV-9 retrovirus is shown on the matrix chart by a continuous line forany partial homology greater than or equal to 70%. It can be seen thatthere are homologies in the flanking regions of the clone (with the polgene at the 5′ end and with the env gene and then the LTR at the 3′ end), but that the internal region is totally divergent and does not displayany homology, even weak, with the env gene of HSERV-9. Furthermore, itis apparent that the clone FBd13 contains a longer “env” region than theone which is described for the defective endogenous HSERV-9; it may thusbe seen that the internal divergent region constitutes an “insert”between the regions of partial homology with the HSERV-9 defectivegenes.

[0331] This additional sequence determines a potential orf, designatedORF B13, which is represented by its amino acid sequence SEQ ID NO:87.

[0332] The molecular structure of the clone FBd13 was analyzed using theGeneWork software and Genebank and SwissProt data banks.

[0333] 5 glycosylation sites were found.

[0334] The protein does not have significant homology with already knownsequences.

[0335] It is probable that this clone originates from a recombination ofan endogenous retroviral element (ERV), linked to the replication ofMSRV-1.

[0336] Such a phenomenon does not lack generation of the expression ofpolypeptides, or even of endogenous retroviral proteins which are notnecessarily tolerated by the immune system. Such a scheme of aberrantexpression of endogenous elements related to MSRV-1 and/or induced bythe latter is liable to multiply the aberrant antigens, and hence tendsto contribute to the induction of autoimmune processes such as areobserved in MS. It clearly constitutes a novel element never hithertodescribed. In effect, interrogation of the data banks of nucleic acidsequences available in version No. 19 (1996) of the “Entrez” software(NCBI, NIH, Bethesda, USA) did not enable a known homologous sequencecomprising the whole of the env region of this clone to be identified.

EXAMPLE 14

[0337] Obtaining a Clone FP6 Containing a Portion of the pol Gene, witha Region Coding for the Reverse Transcriptase Enzyme Homologous to theClone POL* MSRV-1, and a 3′ pol Region Divergent=from the EquivalentSequences Described in the Clones POL*, tpol, FBd3, JLBc1 and JLBc2.

[0338] A 3′ RACE was performed on total RNA extracted from plasma of apatient suffering from MS. A healthy control plasma treated under thesame conditions was used as negative control. The synthesis of cDNA wascarried out with the following modified oligo(dT) primer: 5′ GACTCGCTGCAGATCGATTT TTTTTTTTTT TTTT 3′ (SEQ ID NO:68)

[0339] and Boehringer “Expand RT” reverse transcriptase according to theconditions recommended by the company. A PCR was performed with theenzyme Klentaq (Clontech) under the following conditions: 94° C. 5 minthen 93° C. 1 min, 58° C. 1 min, 68° C. 3 min for 40 cycles and 68° C.for 8 min, and with a final reaction volume of 50 μl.

[0340] Primers used for the PCR:

[0341] 5′ primer, identified by SEQ ID NO:69 5′ GCCATCAAGC CACCCAAGAACTCTTAACTT 3′;

[0342] 3′ primer, identified by SEQ ID NO:68 (=the same as for the cDNA)

[0343] A second, so-called “semi-nested” PCR was carried out with a 5′primer located within the region already amplified. This second PCR wasperformed under the same experimental conditions as those used in thefirst PCR, using 10 μl of the amplification product originating from thefirst PCR.

[0344] Primers used for the semi-nested PCR:

[0345] 5′ primer, identified by SEQ ID NO:70 5′ CCAATAGCCA GACCATTATATACACTAATT 3′;

[0346] 3′ primer, identified by SEQ ID NO:68 (=the same as for the cDNa)Primers SEQ ID NO:69 and SEQ ID NO:70 are specific for the pol* region:position No. 403 to No. 422 and No. 641 to No. 670, respectively.

[0347] An amplification product was thus obtained from the extracellularRNA extracted from the plasma of a patient suffering from MS. Thecorresponding fragment was not observed for the plasma of the healthycontrol. This amplification product was cloned in the following manner.

[0348] The amplified DNA was inserted into a plasmid using the TACloning™ kit. The 2 μl of DNA solution were mixed with 5 μl of steriledistilled water, 1 μl of a 10-fold concentrated ligation buffer “10×LIGATION BUFFER”, 2 μl of “pCR™ VECTOR” (25 ng/ml) and 1 μl of “TA DNALIGASE”. This mixture was incubated overnight at 12° C. The followingsteps were carried out according to the instructions of the TA Cloningkit® (British Biotechnology). At the end of the procedure, the whitecolumns of recombinant bacteria (white) were picked out in order to becultured and to permit extraction of the plasmids incorporated accordingto the so-called “miniprep” procedure (17). The plasmid preparation fromeach recombinant colony was cut with a suitable restriction enzyme andanalyzed on agarose gel. Plasmids possessing an insert detected under UVlight after staining the gel with ethidium bromide was selected forsequencing of the insert, after hybridization with a primercomplementary to the Sp6 promoter present on the cloning plasmid of theTA cloning kit®. The reaction prior to sequencing was then performedaccording to the method recommended for the use of the sequencing kit“Prism ready reaction kit dye deoxyterminator cycle sequencing kit”(Applied Biosystems, ref. 401384), and automatic sequencing was carriedout with an Applied Biosystems “Automatic Sequencer, model 373 A”apparatus according to the manufacturer's instructions.

[0349] The clone obtained, designated FP6, enables a region of 467 bpwhich is 89% homologous to the pol* region of the MSRV-1 retrovirus anda region of 1167 bp which is 64% homologous to the pol region of ERV-9(No. 1634 to 2856) to be defined.

[0350] The clone FP6 is represented in FIG. 38 by its nucleotidesequence identified by SEQ ID NO:61. The three potential reading framesof this clone are indicated by their amino acid sequence under thenucleotide sequence.

EXAMPLE 15

[0351] Obtaining a Region Designated G+E+A Containing an ORF for aRetroviral Protease, by PCR Amplification of the Nucleic Acid SequanceContained Between the 5′ Region Defined by the Clone “GM3” and the 3′Region Defined by the Clone POL*, from the RNA Extracted from a Pool ofPlasmas of Patients Suffering from MS.

[0352] Oligonucleotides specific for the MSRV-1 sequences alreadyidentified by the Applicant were defined in order to amplify theretroviral RNA originating from virions present in the plasma ofpatients suffering from MS. Control reactions were performed so as tomonitor the presence of contaminants (reaction with water). Theamplification consists of a step of RT-PCR followed by a “nested” PCR.Pairs of primers were defined for amplifying three overlapping regions(designated G, E and A) on the regions defined by the sequences of theclones GM3 and pol* described above.

Semi-Nested RT-PCR for Amplification of the Region G:

[0353] in the first RT-PCR cycle, the following primers are used:

[0354] primer 1: SEQ ID NO:71 (sense)

[0355] primer 2: SEQ ID NO:72 (antisense)

[0356] in the second.PCR cycle, the following primers are used:

[0357] primer 1: SEQ ID NO:73 (sense)

[0358] primer 4: SEQ ID NO:74 (antisense)

Nested RT-PCR for Amplification of the Region E:

[0359] in the first RT-PCR cycle, the following primers are used:

[0360] primer 5: SEQ ID NO:75 (sense)

[0361] primer 6: SEQ ID NO:76 (antisense)

[0362] in the second PCR cycle, the following primers are used:

[0363] primer 7: SEQ ID NO:77 (sense)

[0364] primer 8: SEQ ID NO:78 (antisense)

Semi-Nested RT-PCR for Amplification of the Region A:

[0365] in the first RT-PCR cycle, the following primers are used:

[0366] primer 9: SEQ ID NO:79 (sense)

[0367] primer 10: SEQ ID NO:80 (antisense)

[0368] in the second PCR cycle, the following primers are used:

[0369] primer 9: SEQ ID NO:81 (sense)

[0370] primer 11: SEQ ID NO:82 (antisense)

[0371] The primers and the regions G, E and A which they define arepositioned as follows:                                                        cDNA     1    G    4 2              5 7          E           8 6                                         3    A   11 10<-------------------------><-------------------------->               GM3                      POL*

[0372] The sequence of the region defined by the different clones G, Eand A was determined after cloning and sequencing of the “nested”amplification products.

[0373] The clones G, E and A were assembled together by PCR with theprimers 1 at the 5′ end of the fragment G and 11 at the 3′ end of thefragment A, the primers being described above. An approximately 1580-bpfragment G+E+A was amplified and inserted into a plasmid using the TACloning (trademark) kit. The sequence of the amplification productcorresponding to G+E+A was determined and analysis of the G+E and E+Aoverlaps was carried out. The sequence is shown in FIG. 39, andcorresponds to the sequence SEQ ID NO:89.

[0374] A reading frame coding for an MSRV-1 retroviral protease wasfound in the region E. The amino acid sequence of the protease,identified by SEQ ID NO:90, is presented in FIG. 40.

EXAMPLE 16

[0375] Obtaining a Clone LTRGAG12, Related to an Endogenous RetroviralElement (ERV) Close to MSRV-1, in the DNA of an MS Lymphoblastoid LineProducing Virions and Expressing the MSRV-1 Retrovirus.

[0376] A nested PCR was performed on the DNA extracted from alymphoblastoid line (B lymphocytes immortalized with the EBV virusstrain B95, as described above and as is well known to a person skilledin the art) expressing the MSRV-1 retrovirus and originating fromperipheral blood lymphocytes of a patient suffering from MS.

[0377] In the first PCR step, the following primers are used: primerCTCGATTTCT TGCTGGGCCT TA (SEQ ID NO:83) 4327: primer GTTGATTCCCTCCTCAAGCA (SEQ ID NO:84) 3512:

[0378] This step comprises 35 amplification cycles with the followingconditions: 1 min at 94° C., 1 min at 54° C. and 4 min at 72° C.

[0379] In the second PCR step, the following primers are used: primer4294: CTCTACCAAT CAGCATGTGG (SEQ ID NO:85) primer 3591: TGTTCCTCTTGGTCCCTAT (SEQ ID NO:86)

[0380] This step comprises 35 amplification cycles with the followingconditions: 1 min at 94° C., 1 min at 54° C. and 4 min at 72° C.

[0381] The products originating from the PCR were purified afterpurification on agarose gel according to conventional methods (17), andthen resuspended in 10 ml of distilled water. Since one of theproperties of Taq polymerase consists in adding an adenine at the 3′ endof each of the two DNA strands, the DNA obtained was inserted directlyinto a plasmid using the TA Cloning™ kit (British Biotechnology). The 2μl of DNA solution were mixed with 5 μl of sterile distilled water, 1 μlof a 10-fold concentrated ligation buffer “10× LIGATION BUFFER”, 2 μl of“pCR™ VECTOR” (25 ng/ml) and 1 μl of “TA DNA LIGASE”. This mixture wasincubated overnight at 12° C. The following steps were carried outaccording to the instructions of the TA Cloning® kit (BritishBiotechnology). At the end of the procedure, the white colonies ofrecombinant bacteria (white) were picked out in order to be cultured andto permit extraction of the plasmids incorporated according to theso-called “miniprep” procedure (17). The plasmid preparation from eachrecombinant colony was cut with a suitable restriction enzyme andanalyzed on agarose gel. The plasmids possessing an insert detectedunder UV light after staining the gel with ethidium bromide wereselected for sequencing of the insert, after hybridization witn a primercomplementary to the Sp6 promoter present on the cloning plasmid of theTA Cloning Kit®. The reaction prior to sequencing was then performedaccording to the method recommended for the use of the sequencing kit“Prism ready reaction kit dye deoxyterminator cycle. sequencing kit”(Applied Biosystems, ref. 401384), and automatic sequencing was carriedout with an Applied Biosystems “Automatic Sequencer, model 373 A”apparatus according to the manufacturer's instructions.

[0382] Thus, a clone designated LTRGAG12 could be obtained, and isrepresented by its internal sequence identified by SEQ ID NO:60.

[0383] This clone is probably representative of endogenous elementsclose to ERV-9, present in human DNA, in particular in the DNA ofpatients suffering from MS, and capable of interfering with theexpression of the MSRV-1 retrovirus, hence capable of having a role inthe pathogenesis associated with the MSRV-1 retrovirus and capable ofserving as marker for a specific expression in the pathology inquestion.

EXAMPLE 17

[0384] Detection of Anti-MSRV-1 Specific Antibodies in Human Serum.

[0385] Identification of the sequence of the pol gene of the MSRV-1retrovirus and of an open reading frame of this gene enabled the aminoacid sequence SEQ ID NO:63 of a region of the said gene, referenced SEQID NO:62, to be determined.

[0386] Different synthetic peptides corresponding to fragments of theprotein sequence of MSRV-1 reverse transcriptase encoded by the pol genewere tested for their antigenic specificity with respect to sera ofpatients suffering from MS and of healthy controls.

[0387] The peptides were synthesized chemically by solid-phase synthesisaccording to the Merrifield technique (22). The practical details arethose described below.

a) Peptide Synthesis:

[0388] The peptides were synthesized on a phenylacetamidomethyl(PAM)/polystyrene/divinylbenzene resin (Applied Biosystems, Inc. FosterCity, CA), using an “Applied Biosystems 430A” automatic synthesizer. Theamino acids are coupled in the form of hydroxybenzotriazole (HOBT)esters. The amino acids used are obtained from Novabiochem(Läuflerlfingen, Switzerland) or Bachem (Bubendorf, Switzerland).

[0389] The chemical synthesis was performed using a double couplingprotocol with N-methylpyrrolidone (NMP) as solvent. The peptides werecut from the resin, as well as the side-chain protective groups,simultaneously, using hydrofluoric acid (HF) in a suitable apparatus(type I cleavage apparatus, Peptide Instiute, Osaka, Japan).

[0390] For,1 g of peptidyl resin, 10 ml of HF, 1 ml of anisole and 1 mlof dimethyl sulphide SDMS are used. The mixture is stirred for 45minutes at −2° C. The HP is then evaporated off under vacuum. Afterintensive washes with ether, the peptide is eluted from the resin with10% acetic acid and then lyophilized.

[0391] The peptides are purified by preparative high performance liquidchromatography on a VYDAC C18 type column (250×21 mm) (The SeparationGroup, Hesperia, CA, USA). Elution is carried out with an acetonitrilegradient at a flow rate of 22 ml/min. The fractions collected aremonitored by an elution under isocratic conditions on a VYDAC® C18analytical column (250×4.6 mm) at a flow rate of 1 ml/min. Fractionshaving the same retention time are pooled and lyophilized. Thepreponderant fraction is then analysed by analytical high performanceliquid chromatography with the system described above. The peptide whichis considered to be of acceptable purity manifests itself in a singlepeak representing not less than 95% of the chromatogram.

[0392] The purified peptides are then analysed with the object ofmonitoring their amino acid composition, using an Applied Biosystems420H automatic amino acid analyser. Measurement of the (average)chemical molecular mass of the peptides is obtained using LSIMS massspectrometry in the positive ion mode on a VG. ZAB.ZSEQ double focusinginstrument connected to a DEC-VAX 2000 acquisition system (VG analyticalLtd, Manchester, England).

[0393] The reactivity of the different peptides was tested against seraof patients suffering from MS and against sera of healthy controls. Thisenabled a peptide designated S24Q to be selected, whose sequence isidentified by SEQ ID NO:63, encoded by a nucleotide sequence of the polgene of MSRV-1 (SEQ ID NO:62).

b) Antigenic Properties:

[0394] The antigenic properties of the S24Q peptide were demonstratedaccording to the ELISA protocol described below.

[0395] The lyophilized S24Q peptide was dissolved in 10 % acetic acid ata concentration of 1 mg/ml. This stock solution was aliquoted and keptat +4° C. for use over a fortnight, or frozen at −20° C. for use within2 months. An aliquot is diluted in PBS (phosphate buffered saline)solution so as to obtain a final peptide concentration of 5micrograms/ml. 100 microlitres of this dilution are placed in each wellof Nunc Maxisorb (trade name) microtitration plates. The plates arecovered with a “plate-sealer” type adhesive and kept for 2 hours at +37°C. for the phase of adsorption of the peptide to the plastic. Theadhesive is removed and the plates are washed three times with a volumeof 300 microlitres of a solution A (1 ×PBS, 0.05% Tween 20®), theninverted over an absorbent tissue. The plates thus drained are filledwith 250 microlitres per well of a solution B (solution A+10% of goatserum), then covered with an adhesive and incubated for 1 hour at 37° C.The plates are then washed three times with the solution A as describedabove.

[0396] The test serum samples are diluted beforehand to {fraction(1/100)} in the solution B, and 100 microlitres of each dilute testserum are placed in the wells of each microtitration plate. A negativecontrol is placed in one well of each plate, in the form of 100microlitres of buffer B. The plates covered with an adhesive are thenincubated for 1 hour 30 min at 37° C. The plates are then washed threetimes with the solution A as described above. For the IgG response, aperoxidase-labelled goat antibody directed against human IgG (marketedby Jackson Immuno Research Inc.) is diluted in the solution B (dilution{fraction (1/10,000)}). 100 microlitres of the appropriate dilution ofthe labelled antibody are then placed in each well of the microtitrationplates, and the plates covered with an adhesive are incubated for 1 hourat 37° C. A further washing of the plates is then performed as describedabove. In parallel, the peroxidase substrate is prepared according tothe directions of the BioMérieux kits. 100 microlitres of substratesolution are placed in each well, and the plates are placed protectedfrom light for 20 to 30 minutes at room temperature.

[0397] When the colour reaction has stabilized, 50 microlitres of Color2 (BioMérieux trade name) are placed in each well in order to stop thereaction. The plates are placed immediately in an ELISA platespectrophotometric reader, and the optical density (OD) of each well isread at a wavelength of 492 nm.

[0398] The serological samples are introduced in duplicate or intriplicate, and the optical density (OD) corresponding to the serumtested is calculated by taking the mean of the OD values obtained forthe same sample at the same dilution.

[0399] The net OD of each serum corresponds to the mean OD of the serumminus the mean OD of the negative control (solution B: PBS, 0.05% Tween20®, 10% goat serum).

c) Detection of Anti-MSRV-1 IgG Antibodies (S240) by ELISA

[0400] The technique described above was used with the S24Q peptide totest for the presence of anti-MSRV-1 specific IgG antibodies in theserum of 15 patients for whom a definite diagnosis of MS was establishedaccording to the criteria of Poser (23), and of 15 healthy controls(blood donors).

[0401]FIG. 41 shows the results for each serum tested with an anti-IgGantibody. Each vertical bar represents the net optical density (OD at492 nm) of a serum tested. The ordinate axis gives the net OD at the topof the vertical bars. The first 15 vertical bars lying to the left ofthe vertical broken line represent the sera of 15 healthy controls(blood donors), and the 15 vertical bars lying to the right of thevertical broken line represent the sera of 15 cases of MS tested. Thediagram enables 2 controls to be revealed whose OD rises above thegrouped values of the control population. These values may represent thepresence of specific IgGs in symptomless seropositive patients. Twomethods were hence evaluated in order to determine the statisticalthreshold of positivity of the test.

[0402] The mean of the net OD values for the controls, including thecontrols with high net OD values, is 0.129 and the standard deviation is0.06. Without the 2 controls whose OD values are greater than 0.2, themean of the “negative” controls is 0.107 and the standard deviation is0.03. A theoretical threshold of positivity may be calculated accordingto the formula:

[0403] threshold value (mean of the net OD values of the negativecontrols)+(2 or 3×standard deviation of the net OD values of thenegative controls).

[0404] In the first case, there are considered to be symptomlessseropositives, and the threshold value is equal to 0.11+(3×0.03)=0.20.The negative results represent a non-specific “background” of thepresence of antibodies directed specifically against an epitope of thepeptide.

[0405] In the second case, if the set of controls consisting of blooddonors in apparent good health is taken as a reference basis, withoutexcluding the sera which are, on the face of it, seropositive, thestandard deviation of the “non-MS controls” is 0.116. The thresholdvalue then becomes 0.13 +(3×0.06)=0.31.

[0406] According to this latter analysis, the test is specific for MS.In this respect, it is seen that the test is specific for MS, since, asshown in Table 1, no control has a net OD above this threshold. In fact,this result reflects the fact that the antibody titres in patientssuffering from MS are, for the most part, higher than in healthycontrols who have been in contact with MSRV-1.

[0407] In accordance with the first method of calculation, and as shownin FIG. 41 and in Table 3, 6 of the 15 MS sera give a positive result(OD greater than or equal to 0.2), indicating the presence of IgGsspecifically directed against the S24Q peptide, hence against a portionof the reverse transcriptase enzyme of the MSRV-1 retrovirus encoded byits pol gene, and consequently against the MSRV-1 retrovirus.

[0408] Thus, approximately 40% of the MS patients tested have reactedagainst an epitope carried by the S24Q peptide and possess circulatingIgGs directed against the latter.

[0409] Two out of 15 blood donors in apparent good health show apositive result. Thus, it is apparent that approximately 13% of thesymptomless population may have been in contact with an epitope carriedby the S24Q peptide under conditions which have led to an activeimmunization which manifests itself in the persistence of specific serumIgGs. These conditions are compatible with an immunization against theMSRV-1 retrovirus reverse transcriptase during an infection with (and/orreactivation of) the MSRV-1 retrovirus. The absence of apparentneurological pathology recalling MS in these seropositive controls mayindicate that they are healthy carriers and have eliminated aninfectious virus after immunizing themselves, or that they constitute anat-risk population of chronic carriers. In effect, epidemiological datashowing that a pathogenic agent present in the environment of regions ofhigh prevalence of MS may be the cause of this disease imply that afraction of the population free from MS has necessarily been in contactwith such a pathogenic agent. It has been shown that the MSRV-1retrovirus constitutes all or part of this “pathogenic agent” at thesource of MS, and it is hence normal for controls taken from a healthypopulation to possess IgG type antibodies against components of theMSRV-1 retrovirus.

[0410] Lastly, the detection of anti-S24Q antibodies in only one out oftwo MS cases tested here may reflect the fact that this peptide does notrepresent an immunodominant MSRV-1 epitope, that inter-individual strainvariations may induce an immunization against a divergent peptide motifin the same region, or that the course of the disease and the treatmentsfollowed may modulate over time the antibody response against the S24Qpeptide. TABLE NO. 3 CONTROLS MS 0.101 0.136 0.058 0.391 0.126 0.370.131 0.119 0.105 0.267 0.294 0.141 0.116 0.102 0.088 0.18 0.105 0.4110.172 0.164 0.137 0.049 0.223 0 644 0.08 0.268 0.073 0.065 0.132 0.074Mean 0.129 Standard Deviation 0.06 Threshold 0.31

d) Detection of Anti-MSRV-1 IgM Antibodies by ELISA

[0411] The ELISA technique with the S24Q peptide was used to test forthe presence of anti-MSRV-1 IgM specific antibodies in the same sera asabove.

[0412]FIG. 42 shows the results for each serum tested with an anti-IgMantibody. Each vertical bar represents the net optical density (OD at492 nm) of a serum tested. The ordinate axis gives the net OD at the topof the vertical bars. The first 15 vertical bars lying to the left ofthe vertical line cutting the abscissa axis represent the sera of 15healthy controls (blood donors), and the vertical bars lying to theright of the vertical broken line represent the sera of 15 cases of MStested.

[0413] The mean of the OD values for the MS cases tested is 1.6.

[0414] The mean of the net OD values for the controls is 0.7.

[0415] The standard deviation of the negative controls is 0.6.

[0416] The threshold of theoretical positivity may be calculatedaccording to the formula:

[0417] threshold value=(mean of the OD values of the negativecontrols)+(3×standard deviation of the OD values of the negativecontrols).

[0418] The threshold value is hence equal to 0.7+(3×0.6)=2.5;

[0419] The negative results represent a non-specific “background” of thepresence of antibodies directed specifically against an epitope of thepeptide.

[0420] According to this analysis, and as shown in FIG. 42 and in thecorresponding Table 4, the IgM test is specific for MS, since no controlhas a net OD above the threshold. 6 of the 15 MS sera produce a positiveIgM result

[0421] The difference in seroprevalence between the MS and controlpopulations is extremely significant: “chi-squared” test, p <0.002.

[0422] These results point to an aetiopathogenic role of MSRV-1 in MS.

[0423] Thus, the detection of IgM and IgG antibodies against the S24Qpeptide makes it possible to evaluate, alone or in combination withother MSRV-1 peptides, the course of an MSRV-1 infection and/or of theviral reactivation of MSRV-1. TABLE NO. 4 CONTROLS MS 1,449 0,974 0,3716,117 0,448 2,883 0,456 1,945 0,885 1,787 2,235 0,273 0,301 1,766 0,1380,668 0,16 2,603 1,073 0,802 1,366 0,245 0,283 0,147 0,262 2,441 0,5850,287 0,356 0,589 Mean 0,7 Standard Deviation 0,6 Threshold 2,5

[0424] It is possible, as a result of the new discoveries made and thenew methods developed by the inventors, to permit the improvedimplementation of diagnostic tests for MSRV-1 infection and/orreactivation and to evaluate a therapy in MS and/or RA on the basis ofits efficacy in “negativing” the detection of these agents in thepatient's biological fluids. Furthermore, early detection in individualsnot yet displaying neurological signs of MS or rheumatological signs ofRA could make it possible to institute a treatment which would be allthe more effective with respect to the subsequent clinical course forthe fact that it would precede the lesion stage which corresponds to theonset of the clinical disorders. Now, at the present time, a diagnosisof MS or RA cannot be established before a symptomatology of lesions hasset in, and hence no treatment is instituted before the emergence of aclinical picture suggestive of lesions which are already significant.The diagnosis of an MSRV-1 and/or MSRV-2 infection and/or reactivationin man is hence of decisive importance, and the present inventionprovides the means of doing this.

[0425] It is thus possible, apart from carrying out a diagnosis ofMSRV-1 infection and/or reactivation, to evaluate a therapy in MS on thebasis of its efficacy in “negativing” the detection of these agents inthe patients' biological fluids.

BIBLIOGRAPHY

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[0452] 147(12): 4360-4365, 1991.

1 92 1158 base pairs nucleotide single linear cDNA 1 CCCTTTGCCACTACATCAAT TTTAGGAGTA AGGAAACCCA ACGGACAGTG GAGGTTAGTG 60 CAAGAACTCAGGATTATCAA TGAGGCTGTT GTTCCTCTAT ACCCAGCTGT ACCTAACCCT 120 TATACAGTGCTTTCCCAAAT ACCAGAGGAA GCAGAGTGGT TTACAGTCCT GGACCTTAAG 180 GATGCCTTTTTCTGCATCCC TGTACGTCCT GACTCTCAAT TCTTGTTTGC CTTTGAAGAT 240 CCTTTGAACCCAACGTCTCA ACTCACCTGG ACTGTTTTAC CCCAAGGGTT CAGGGATAGC 300 CCCCATCTATTTGGCCAGGC ATTAGCCCAA GACTTGAGTC AATTCTCATA CCTGGACACT 360 CTTGTCCTTCAGTACATGGA TGATTTACTT TTAGTCGCCC GTTCAGAAAC CTTGTGCCAT 420 CAAGCCACCCAAGAACTCTT AACTTTCCTC ACTACCTGTG GCTACAAGGT TTCCAAACCA 480 AAGGCTCGGCTCTGCTCACA GGAGATTAGA TACTNAGGGC TAAAATTATC CAAAGGCACC 540 AGGGCCCTCAGTGAGGAACG TATCCAGCCT ATACTGGCTT ATCCTCATCC CAAAACCCTA 600 AAGCAACTAAGAGGGTTCCT TGGCATAACA GGTTTCTGCC GAAAACAGAT TCCCAGGTAC 660 ASCCCAATAGCCAGACCATT ATATACACTA ATTANGGAAA CTCAGAAAGC CAATACCTAT 720 TTAGTAAGATGGACACCTAC AGAAGTGGCT TTCCAGGCCC TAAAGAAGGC CCTAACCCAA 780 GCCCCAGTGTTCAGCTTGCC AACAGGGCAA GATTTTTCTT TATATGCCAC AGAAAAAACA 840 GGAATAGCTCTAGGAGTCCT TACGCAGGTC TCAGGGATGA GCTTGCAACC CGTGGTATAC 900 CTGAGTAAGGAAATTGATGT AGTGGCAAAG GGTTGGCCTC ATNGTTTATG GGTAATGGNG 960 GCAGTAGCAGTCTNAGTATC TGAAGCAGTT AAAATAATAC AGGGAAGAGA TCTTNCTGTG 1020 TGGACATCTCATGATGTGAA CGGCATACTC ACTGCTAAAG GAGACTTGTG GTTGTCAGAC 1080 AACCATTTACTTAANTATCA GGCTCTATTA CTTGAAGAGC CAGTGCTGNG ACTGCGCACT 1140 TGTGCAACTCTTAAACCC 1158 297 base pairs nucleotide single linear cDNA 2 CCCTTTGCCACTACATCAAT TTTAGGAGTA AGGAAACCCA ACGGACAGTG GAGGTTAGTG 60 CAAGAACTCAGGATTATCAA TGAGGCTGTT GTTCCTCTAT ACCCAGCTGT ACCTAACCCT 120 TATACAGTGCTTTCCCAAAT ACCAGAGGAA GCAGAGTGGT TTACAGTCCT GGACCTTAAG 180 GATGCCTTTTTCTGCATCCC TGTACGTCCT GACTCTCAAT TCTTGTTTGC CTTTGAAGAT 240 CCTTTGAACCCAACGTCTCA ACTCACCTGG ACTGTTTTAC CCCAAGGGTT CAAGGGA 297 85 base pairsnucleotide single linear cDNA 3 GTTTAGGGAT ANCCCTCATC TCTTTGGTCAGGTACTGGCC CAAGATCTAG GCCACTTCTC 60 AGGTCCAGSN ACTCTGTYCC TTCAG 85 86base pairs nucleotide single linear cDNA 4 GTTCAGGGAT AGCCCCCATCTATTTGGCCA GGCACTAGCT CAATACTTGA GCCAGTTCTC 60 ATACCTGGAC AYTCTYGTCCTTCGGT 86 85 base pairs nucleotide single linear cDNA 5 GTTCARRGATAGCCCCCATC TATTTGGCCW RGYATTAGCC CAAGACTTGA GYCAATTCTC 60 ATACCTGGACACTCTTGTCC TTYRG 85 85 base pairs nucleotide single linear cDNA 6GTTCAGGGAT AGCTCCCATC TATTTGGCCT GGCATTAACC CGAGACTTAA GCCAGTTCTY 60ATACGTGGAC ACTCTTGTCC TTTGG 85 111 base pairs nucleotide single linearcDNA 7 GTGTTGCCAC AGGGGTTTAR RGATANCYCY CATCTMTTTG GYCWRGYAYT RRCYCRAKAY60 YTRRGYCAVT TCTYAKRYSY RGSNAYTCTB KYCCTTYRGT ACATGGATGA C 111 645 basepairs nucleotide single linear cDNA 8 TCAGGGATAG CCCCCATCTA TTTGGCCAGGCATTAGCCCA AGACTTGAGT CAATTCTCAT 60 ACCTGGACAC TCTTGTCCTT CAGTACATGGATGATTTACT TTTAGTCGCC CGTTCAGAAA 120 CCTTGTGCCA TCAAGCCACC CAAGAACTCTTAACTTTCCT CACTACCTGT GGCTACAAGG 180 TTTCCAAACC AAAGGCTCGG CTCTGCTCACAGGAGATTAG ATACTNAGGG CTAAAATTAT 240 CCAAAGGCAC CAGGGCCCTC AGTGAGGAACGTATCCAGCC TATACTGGCT TATCCTCATC 300 CCAAAACCCT AAAGCAACTA AGAGGGTTCCTTGGCATAAC AGGTTTCTGC CGAAAACAGA 360 TTCCCAGGTA CASCCCAATA GCCAGACCATTATATACACT AATTANGGAA ACTCAGAAAG 420 CCAATACCTA TTTAGTAAGA TGGACACCTACAGAAGTGGC TTTCCAGGCC CTAAAGAAGG 480 CCCTAACCCA AGCCCCAGTG TTCAGCTTGCCAACAGGGCA AGATTTTTCT TTATATGCCA 540 CAGAAAAAAC AGGAATAGCT CTAGGAGTCCTTACGCAGGT CTCAGGGATG AGCTTGCAAC 600 CCGTGGTATA CCTGAGTAAG GAAATTGATGTAGTGGCAAA GGGTT 645 741 base pairs nucleotide single linear cDNA 9CAAGCCACCC AAGAACTCTT AAATTTCCTC ACTACCTGTG GCTACAAGGT TTCCAAACCA 60AAGGCTCAGC TCTGCTCACA GGAGATTAGA TACTTAGGGT TAAAATTATC CAAAGGCACC 120AGGGGCCTCA GTGAGGAACG TATCCAGCCT ATACTGGGTT ATCCTCATCC CAAAACCCTA 180AAGCAACTAA GAGGGTTCCT TAGCATGATC AGGTTTCTGC CGAAAACAAG ATTCCCAGGT 240ACAACCAAAA TAGCCAGACC ATTATATACA CTAATTAAGG AAACTCAGAA AGCCAATACC 300TATTTAGTAA GATGGACACC TAAACAGAAG GCTTTCCAGG CCCTAAAGAA GGCCCTAACC 360CAAGCCCCAG TGTTCAGCTT GCCAACAGGG CAAGATTTTT CTTTATATGG CACAGAAAAA 420ACAGGAATCG CTCTAGGAGT CCTTACACAG GTCCGAGGGA TGAGCTTGCA ACCCGTGGCA 480TACCTGAATA AGGAAATTGA TGTAGTGGCA AAGGGTTGGC CTCATNGTTT ATGGGTAATG 540GNGGCAGTAG CAGTCTNAGT ATCTGAAGCA GTTAAAATAA TACAGGGAAG AGATCTTNCT 600GTGTGGACAT CTCATGATGT GAACGGCATA CTCACTGCTA AAGGAGACTT GTGGTTGTCA 660GACAACCATT TACTTAANTA TCAGGCTCTA TTACTTGAAG AGCCAGTGCT GNGACTGCGC 720ACTTGTGCAA CTCTTAAACC C 741 93 base pairs nucleotide single linear cDNA10 TGGAAAGTGT TGCCACAGGG CGCTGAAGCC TATCGCGTGC AGTTGCCGGA TGCCGCCTAT 60AGCCTCTACA TGGATGACAT CCTGCTGGCC TCC 93 96 base pairs nucleotide singlelinear cDNA 11 TTGGATCCAG TGYTGCCACA GGGCGCTGAA GCCTATCGCG TGCAGTTGCCGGATGCCGCC 60 TATAGCCTCT ACGTGGATGA CCTSCTGAAG CTTGAG 96 748 base pairsnucleotide single linear cDNA 12 TGCAAGCTTC ACCGCTTGCT GGATGTAGGCCTCAGTACCG GNGTGCCCCG CGCGCTGTAG 60 TTCGATGTAG AAAGCGCCCG GAAACACGCGGGACCAATGC GTCGCCAGCT TGCGCGCCAG 120 CGCCTCGTTG CCATTGGCCA GCGCCACGCCGATATCACCC GCCATGGCGC CGGAGAGCGC 180 CAGCAGACCG GCGGCCAGCG GCGCATTCTCAACGCCGGGC TCGTCGAACC ATTCGGGGGC 240 GATTTCCGCA CGACCGCGAT GCTGGTTGGAGAGCCAGGCC CTGGCCAGCA ACTGGCACAG 300 GTTCAGGTAA CCCTGCTTGT CCCGCACCAACAGCAGCAGG CGGGTCGGCT TGTCGCGCTC 360 GTCGTGATTG GTGATCCACA CGTCAGCCCCGACGATGGGC TTCACGCCCT TGCCACGCGC 420 TTCCTTGTAG ANGCGCACCA GCCCGAAGGCATTGGCGAGA TCGGTCAGCG CCAAGGCGCC 480 CATGCCATCT TTGGCGGCAG CCTTGACGGCATCGTCGAGA CGGACATTGC CATCGACGAC 540 GGAATATTCG GAGTGGAGAC GGAGGTGGACGAAGCGCGGC GAATTCATCC GCGTATTGTA 600 ACGGGTGACA CCTTCCGCAA AGCATTCCGGACGTGCCCGA TTGACCCGGA GCAACCCCGC 660 ACGGCTGCGC GGGCAGTTAT AATTTCGGCTTACGAATCAA CGGGTTACCC CAGGGCGCTG 720 AAGCCTATCG CGTGCAGTTG CCGGATGC 74818 base pairs nucleotide single linear cDNA 13 GCATCCGGCA ACTGCACG 18 20base pairs nucleotide single linear cDNA 14 GTAGTTCGAT GTAGAAAGCG 20 18base pairs nucleotide single linear cDNA 15 GCATCCGGCA ACTGCACG 18 23base pairs nucleotide single linear cDNA 16 AGGAGTAAGG AAACCCAACG GAC 2319 base pairs nucleotide single linear cDNA 17 TAAGAGTTGC ACAAGTGCG 1921 base pairs nucleotide single linear cDNA 18 TCAGGGATAG CCCCCATCTA T21 24 base pairs nucleotide single linear cDNA 19 AACCCTTTGC CACTACATCAATTT 24 15 base pairs nucleotide single linear cDNA 5, 7, 10, 13 Nrepresents inosine (i) 20 GGTCNTNCCN CANGG 15 21 base pairs nucleotidesingle linear cDNA 21 TTAGGGATAG CCCTCATCTC T 21 21 base pairsnucleotide single linear cDNA 22 TCAGGGATAG CCCCCATCTA T 21 24 basepairs nucleotide single linear cDNA 23 AACCCTTTGC CACTACATCA ATTT 24 23base pairs nucleotide single linear cDNA 24 GCGTAAGGAC TCCTAGAGCT ATT 2318 base pairs nucleotide single linear cDNA 25 TCATCCATGT ACCGAAGG 18 20base pairs nucleotide single linear cDNA 26 ATGGGGTTCC CAAGTTCCCT 20 20base pairs nucleotide single linear cDNA 27 GCCGATATCA CCCGCCATGG 20 18base pairs nucleotide single linear cDNA 28 GCATCCGGCA ACTGCACG 18 20base pairs nucleotide single linear cDNA 29 CGCGATGCTG GTTGGAGAGC 20 20base pairs nucleotide single linear cDNA 30 TCTCCACTCC GAATATTCCG 20 26base pairs nucleotide single linear cDNA 31 GATCTAGGCC ACTTCTCAGG TCCAGS26 23 base pairs nucleotide single linear cDNA 6, 12, 19 N representsinosine (i) 32 CATCTNTTTG GNCAGGCANT AGC 23 24 base pairs nucleotidesingle linear cDNA 33 CTTGAGCCAG TTCTCATACC TGGA 24 22 base pairsnucleotide single linear cDNA 34 AGTGYTRCCM CARGGCGCTG AA 22 22 basepairs nucleotide single linear cDNA 35 GMGGCCAGCA GSAKGTCATC CA 22 22base pairs nucleotide single linear cDNA 36 GGATGCCGCC TATAGCCTCT AC 2222 base pairs nucleotide single linear cDNA 37 AAGCCTATCG CGTGCAGTTG CC22 40 base pairs nucleotide single linear cDNA 38 TAAAGATCTA GAATTCGGCTATAGGCGGCA TCCGGCAAGT 40 50 amino acids amino acid linear peptide 39 AspAla Phe Phe Cys Ile Pro Val Arg Pro Asp Ser Gln Phe Leu Phe 1 5 10 15Ala Phe Glu Asp Pro Leu Asn Pro Thr Ser Gln Leu Thr Trp Thr Val 20 25 30Leu Pro Gln Gly Phe Arg Asp Ser Pro His Leu Phe Gly Gln Ala Leu 35 40 45Ala Gln 50 299 base pairs nucleic acid single linear cDNA 40 GATGCCTTTTTCTGCATCCC TGTACGTCCT GACTCTCAAT TCTTGTTTGC CTTTGAAGAT 60 CCTTTGAACCCAACGTCTCA ACTCACCTGG ACTGTTTTAC CCCAAGGGTT CAGGGATAGC 120 CCCATCTATTTGGCCAGGCA TTAGCCCAAG ATGCCTTTTG CATCCCTGTA CGTGACTCTC 180 AATTCTTGTTTGCCTTTGCC TTTGAAGATG CTTTGAACCC AACGTCTCAA CTCACCTGGA 240 CTGTTTTACGCCAAGGGTTC AGGGATAGCC CCCATCTATT TGGCCAGGCA TTAGCCCAA 299 11 amino acidsamino acid linear peptide 41 Cys Ile Pro Val Arg Pro Asp Ser Gln Phe Leu1 5 10 17 amino acids amino acid linear peptide 42 Val Leu Pro Gln GlyPhe Arg Asp Ser Pro His Leu Phe Gly Glu Ala 1 5 10 15 Leu 17 8 aminoacid amino acid linear peptide 43 Leu Phe Ala Phe Glu Asp Pro Leu 1 5 88 amino acids amino acid linear peptide 44 Phe Ala Phe Glu Asp Pro LeuAsn 1 5 8 25 base pairs nucleic acid single linear cDNA 45 GTGCTGATTGGTGTATTTAC AATCC 25 1859 base pairs nucleic acid single linear cDNA 46GTGCTGATTG GTGTATTTAC AATCCTTTAT CTAATCCGAA ATGCCCATGT TGCAATATGG 60AAAGAAAGGG AGTTCCTAAC CTCTGGGGGA ACCCCCATTA AATACCACAA GTAAATCATG 120GAGTTATTGC ACACAGTGCA AAAACTCAAG GAGGTGGAAG TCTTACACTG CCAAAGCCAT 180CAGAAAAGGG AAGAGGGGAG AAGAGCAGCA TAAGTGGCTA CAGAGGCAAG GAAAGACTAG 240CAGAAAGGAA AGAGAGAAAG AGACAGAAAG TCAGAGAGAG AGAGAGGAAG AGACAGAGCA 300CAAAGAGGGA GTCAGAGAGA GAGAGAGACA GAGAGTCAGA GAGAAGGAAA GAGAGAGAGG 360AAGAGACAAA GAATGAATCA AACAGAGAGA CAGAAAGTCA GAGAGAGAGA GAGAGAGGAA 420GAGACAGAGA AAAAGAGGGA GTCAGAAAAA GAGAGACCAA AGAAGAAGTC CAAAGAGAAA 480GAAAGAGAGA TGGAAGTAGT AAAGGAAAAA CAGTGTACCC TATTCCTTTA AAAGCCGGGG 540TAAATTTAAA ACCTATAATT GATAACTGAA GGTCTTCTCT GTAACCCTGT AACACTCCAA 600TACCACCTTG TTGTCAAGTG TAAACAAGGG CGTAGCCCAA AAGCACTGAG GCCACTAACA 660ACCCATAGCC TTCCTATCAA AATTCCTTAA CCCAGCAGGT TTCCTAACAG GGGATCTAAA 720TCTTAATTAA TTACCATACA ATGGTCCAAC CAGACTTAGG AGGAATTCCC TTCAGGACGG 780GAAGATAGAT GCTTCCTCCC AGGCGATTAA GGGAGAAAGA CACAATGGGT ATTCAGTAAG 840TGCCAAGGGG AACACTTGTA GAAGCAAAGT TAGGAAAATT GCCAAATAAT TGGTTTGCTC 900AAGAGTTGTT TGCACTCAGC CAAACCTTGA AGTACTTGCA GAATCAGAAA GGAGCCATCT 960ATACCAATTC TAAGTTAATA TGGACTGAAG GAGGTTTTAT TAATACCAAA GAGAAATTAA 1020AATCCCAAAC TTATAAGGTT TTCAACCAAA GTAAAGTTTG CTAAAAGTTA ACAGCGTAAC 1080ATGTATTATC CTACTACCAC ACACTCTCAA AGGATTTCTC AGACAGTTTG CAAGAAATAA 1140TGATATCTAT CCTTACTCTA CAATCCCAAA TAGACTCTTT GGCAGCAGTG ACTCTCCAAA 1200ACCGTCAAGG CCTAGACCTC CTCACTGCTG AGAAAGGAGG ACTCTGCACC TTCTTAAGGG 1260AAGAGTGTTG TCTTTACACT AACCAGTCAG GGATAGTATG AGATGCTGCC CGGCATTTAC 1320AGAAAAAGGC TTCTGAAATC AGACAACGCC TTTCAAATTC CTATACCAAC CTCTGGAGTT 1380GGGCAACATG GTTTCTTCCC TTTCTATGTC CCATGGCTGC CATCTTGCTA TTACTCGCCT 1440TTGGGCCCTG TATTTTTAAC CTCCTTGTCA AATTTGTTTC TTCTAGGATC GAGGCCATCA 1500AGCTACAGAT GGTCTTACAA ATGGAACCCC AAATGAGCTC AACTATCAAC TTCTACTGAG 1560GACCCCTAGA CCAACCCCCT GGCCCTTTCA CTGGCCTAAA GAGTTCCCCT CTGGAGGACA 1620CTACCACTGC AGGGCCCCAT CTTTGCCCCT ATCCAGAAGG AAGTAGCTAG AGCAGTCATT 1680GCCCAATTCC CAAGAGCAGC TGGGGTGTCC CGTTTAGAGT GGGGATTGAG AGGTGAAGCC 1740AGCTGGACTT CTGGGTCGGG TGGGGACTTG GAGAACTTTT GTGTCTAGCT AAAGGATTGT 1800AAATGCAACA ATCAGTGCTC TGTGTCTAGC TAAAGGATTG TAAATACACC AATCAGCAC 1859 23base pairs nucleic acid single linear cDNA 47 TGATGTGAAC GGCATACTCA CTG23 24 base pairs nucleic acid single linear cDNA 48 CCCAGAGGTTAGGAACTCCC TTTC 24 25 base pairs nucleic acid single linear cDNA 49GCTAAAGGAG ACTTGTGGTT GTCAG 25 22 base pairs nucleotide single linearcDNA 50 CAACATGGGC ATTTCGGATT AG 22 400 base pairs nucleotide singlelinear cDNA 51 GGCTGCTAAA GGAGACTTGT GGTTGTCAGA CAATCGCCTA CTTAGGTACCAGGCCTTATT 60 ACTTGAGGGA CTGGTGCTTC AGATGCGCAC TTGTGCAGCT CTTAACCCAAACTTATGCTG 120 CCCAGAAGGA TCTTTTAGAG GTCCCCTTAG CCAACCCTGA CCTCAACCTATATATATACT 180 GATGGAAGTT CGTTTGTAGA AAAGGGATTA CAAAGGGNAG GATATNCCATAGGTTAGTGA 240 TAAAGCAGTA CTTGAAAGTA AGCCTCTTCC CCCCAGGGAC CAGCGCCCCCGTTAGCAGAA 300 CTAGTGGCAC TGACCCCGAG CCTTAGAACT TGGAAAGGGA GGAGGATAAATGTGTATACA 360 GATAGCAAGT ATGCTTATCT AATCCGAAAT GCCCATGTTG 400 2389 basepairs nucleotide single linear cDNA 52 TCAGGGATAG CCCCCATCTA TTTGGTCAGGCACTGGCCCA AGATCTAGGG ACATGCCACT 60 TTTAAGAGCC ATTTCTCAAG TCCAGGTACTCTGGTCCTTC GGTATGTGGA TGATTTACTT 120 TTGGCTACCA GTTCAGTAGC CTCATGCCAGCAGGCTACTC TAGATCTCTT GAACTTTCTA 180 GCTAATCAAG GGTACAAGGC ATCTAGGTTGAAGGCCCAGC TTTGCCTACA GCAGGTCAAA 240 TATCTAGGCC TAATCTTAGC CAGAGGGACCAGGGCACTCA GCAAGGAACA AATACAGCCT 300 ATACTGGCTT ATCCTCACCC TAAGACATTAAAACAGTTGC GGGGGTTCCT TGGAATCACT 360 GGCTTTTTGG TGACTATGGA TTCCCAGATACAGCAAGATT GGCAGGCCCC TCTATACTGT 420 AATCAAGGAG ACTCACGAGG GCAAGTACTCATCTAGTAGA ATGGGAACTA GGGACAGAAA 480 CAGCCTTCAA AACCTTAAAG CAGGCCCTAGTACAATCTCC AGCTTTAAGC CTTCCCACAG 540 GACAAAACTT CTCTTTATAC ATCACAGAGAGGGCAGAGAT AGCTCTTGGT GTCCTTATTC 600 AGACTCATGG GACTACCCCA CAACCAGTGGCACACCTAAG TAAGGAAATT GATGTAGTAG 660 CAAAAGGCTG GCCTCACTGT TTATGGGTAGCTGTGGTGGT GGCTGTCTTA GTGTCAGAAG 720 CTATCAAAAT AATACAAGGA AAGGATCTCACTGTCTGGAC TACTCATGAT GTAATGGCAT 780 ACTAGGTGCC AAAAGAAGTT TATGGGTATCAGACAACCAC CTGCTTAGAT ACCAGGGACT 840 ACTCCTGGAG GATTGGGCTT CAAGTGCGTTTTTTGTGGCC TCAACCCTGC CACTTTTCCT 900 CCAGAGGATG GAGAGCCGCT TGAGCATGCTTGCCAACAGG TTGTAGGCCA GAATTATTCC 960 ACCCGAGATG ATCTCTTAGA GTACCCTTAGCTAATCCTGA CCTTAACCTA TATACCAATG 1020 GAAGTTCATT TGTGGAAAAC GGGATATGAAGGGCAGGTTA TGTCATAGTT AGTGATGTAA 1080 TCATACTTGC AAGTAAGCCT CTTACCCCAGGGGCCAGCAC TCAGTTAGCA GAACTAGTCA 1140 CACTTACCTT AACCTTAGAA CTGGGAAAGGGAAAAAGAAT AAATATGTAT ACAGATAGTA 1200 AGTATGCTTA TCTAATCCTA CATGCCCATGCTGCAATATG GAAGGAAAGG GAGTTCCTAA 1260 CCCCTGGGGG AACCCCCATT AAATACCACAAGGYAAATCA TGGAGTTATT GCACGCAGTG 1320 CAAAAACTCA AGGAGGTGGC AGTCTTACACTGCCGAAGCY ATCAAAAAGG GGAAGGAGAG 1380 GGGAGAACAG CAGCATAAGT GGTTGGCAGAGGCAGTGAAA GACCAGCAGA GAGAAGGAGA 1440 GAGACAACGT CAACGACAGA AGGAAAGAAGAGGAGGAGAC AGAGAGGAAG AGACAGAGAG 1500 ACAGTTAGTC CAAGAGAGAG ACAGAGAGAGGAAGAGACAG ACAGAAAGTC CAAGAGAGAA 1560 GGAAAGAGAG GAAGAGACCA AGGAGTCCNAGAGAGAGAAA GAGATAGAAG TAGTAAAGAA 1620 AAAACATTGT ACCCTATTCC TTTAAAAGCCGGGGTATATT TAAAACCTAT AATTGATAAT 1680 TGAGTTCTTG CACCCTCCTC CAGGGGATYGCTGGGAGGAA ACCCTCAACC GATATGTGAA 1740 AATTGTGGGT CGTCCCTATG TCTCAATTACCAGCCAATAC CCCCTTGTTT TTAGTGTGAA 1800 CGAGGGTGTA GAGCGCAGAC AGGGAGACCTCTGACAATCC ATACCCTTCC TATCCAAAAT 1860 CCTTAACCCA GCAGGTTTTC TAAAAGGGGATCTAAATCTT AATTAATTAC CATACAAAGG 1920 TCAAACCAGA TCTAGGAGGA ACTTCCTTCAGGACAGGATG ATAGATGGTT CCTCCCAGGC 1980 GATTAAAGAA AATAAAAAGA CACATGGGCAGCCAGTAAGT GATAAGGGAA CACTAGTAGA 2040 AGCAGTTAGG AGAAGTTGCC TAATAATTGGTCTACTCCAA ATGTGTGAGT TGTTCGCACT 2100 CAGCCCAAAT CTTAAAGTAC TTACAGAATTAGGGAGGAGC CATTTACACC AATTCTAAGT 2160 TAATATGGAC TGGATGAGGT TTTATTAATAGCGAAGGAGA ATTAAATCCT AAACTNACAA 2220 GGTTTTCAAC TAAAGTAAAT TTTACTAAAAGCTAACAGTG TAACATGCAT TATCCTACTA 2280 CAACACACTC TCANAGGATT CCTCAGACAGTTTACAAGAA ATAACAAAAT CTATCTGGTA 2340 AGGATAGTAA CTACAATCCC AAATACATTCTTTGGCAGCA GTGACTCTC 2389 2448 base pairs nucleotide single linear cDNA53 TCAGGGATAG CCCCCATCTA TTTGATCAGG CACTAGCCCA AGATCTAGGC CACTTCTGAA 60GTCCAGGCAT TCTAGTCCTT CAGTATGTGG ATGATTTACT TTTGGCTACC AGTTTGGAAG 120CCTCATGCCA GCAGGCTACT TGAGATCTCT TGAACTTTCT AGCTAATCAA GGGTGTATGG 180CATCTAAATT GAAAGTCCAG CTCTGCCTAC AACAAGTCAA ATATCTAGGC CTAATCTTAG 240ATAGAAGAAC CAGGGCCCTC AGCAAGGAAT GAATAAAGCC TATGCTGGCT TATCGGCACC 300CTAAGACATT AAAACAATTG TGGGGGTTCC TTGGAATCAC TGGCTTTTGC CGACTATGGA 360TCCCTGGATA GAGTGAGATA GCCAGGCCCC CTCTATTACT CTTATCAAGG AGACCCAGAG 420GGCAAATACT TATCTAGTAT TATGGGNACC AGAGGCAGAA AAAGCCTTCC AAACCTTAAA 480GGAGACCCTA GTACAAGCTC CAGCTTTAAG CCTTCCCACA GGACAAANCT TCTCTTTATA 540TGTCACAGAG AGAGCAGGAA TAGCTCCTGG AGTCCTTACT CAGACTTTTG GACGACCCCA 600CGGCCAGTGG CRTACCTAAG TAAGGAAATT GATGTAGTAG CAAAAGGCTG GCCTCACTGT 660TTATGGGTAG TTGCGGCTGT GGCAGTCTTA CTGTCAAAGG CTATCAAAAT AATACAAGGA 720AAGGATTTCA CTATCTGGAC TACTCATGAG GAAAATGGCA TATTAGGTGC CAAAGGAAGT 780TTTTGGCTAT CAGACAACCA CCTGCTCAGA TTCCAGGCAC TACTGATTGA GAGACCAGTG 840CTTTAAATAT GTATGTGTGT GTGTGGCCCT CAACCCTGCC ACTGTTCTCC CAGAAGATGG 900AGAACCAATG AAGCATTACT GTCAACAAAT TAGAGTCCAG AGTTATGCTG CCTGAGAGGA 960TCTCTTAGAA GTCCCCTTAG CTAATCCTGA CCTTAACCTA TATGCTGATG GAAGTTCACT 1020TGTGGAGAAT GGGATACGAA AAGCACATTA TGCCATAGTT AGTGAGGTAA CAGTACTTGA 1080AAGTAAGCCT ATTCCCCCAT GGACCAGAGC CCAGTTAGCA GAACTAGTGG CACTTACCCA 1140AGCCTTAGAA CTAGGAAAGG GAAAAATAAT AAATGTGTAT ACAGATAGCA AGTATGCTTA 1200TCTAATCCTA CATGCCCATG CTGCAGTATG GAAAGAAAGG GAGTTCCTAA CCTCTGGGGG 1260AACCCCCATT AAATACCACA AGGCAAATCA TGGAGTTATT GCATGTAGTG CAAAACCTCA 1320AGTAGGTGGC AGTTTTACAC TGCCTGAAGC TATGGGGAAG GAGAGAGGAG AACAGCAGCA 1380TAAGTGGCTA GCAGAGGCAG CGAAAGACTA GCAGAGAGGA GAGGTAGGGG AAAGACAGAA 1440AGTCAAAGAA AAGAAGTCAA AGACAGACAG AGAAAGAGAC AGAGGGAGCC AGAGAGAAAG 1500AAAAGAGAGA ACGAAAGAGA CAGAATGTCA AAGAACAGAA GAGAGAGGCA GCGCCAGAAG 1560AGTTAAGAAA GTGAGAAAGA GAGATGGAAA TAGTAAAGAA AAAACAGTGT ACCCTATTCC 1620TTTAAAAGCC AGGGTAAATT TAAAACGTAT AATTTTATAA TTGGAAGGTC TTCTCCATAA 1680CCCTATAACA TTAAAATACC ACCTTGTTGT CAGTGTAAAC AAGAGCATAG CCCAAAAGCA 1740CTGAGGCCAC TGACAACCCA TAGCCTTCCT ATCAAAAATC CTTAACTCTG CAGGTTTCCT 1800AACAGGGGAT CTAAATCTCA ACTAATCACC ATACAATGGT CCGACCAGAC CTAGGAGCGA 1860CTCCCCTCAG GACAGAAGGA TGGATGGTTC CTCCCAGGCC ATTAAGGGAA AGAGACACAA 1920TGGGTATTCA GTAAGTGATA AGGGAACTCT TGTAGAAGCA GTTAGGAAGA TTGCCTAATA 1980TTTGGTCTGC TCAAATGTGC CAGCTGTTTG CACTCAGCTA AACCTTAAAT TACTTACAGA 2040ATTAGGAAGG AGCCATCTAT ACCAATTCTG AGTTAATATG AGCTGAACAA GTTCTTATTA 2100ATAGCAAAGA ATCATTGAAA TCTCAAACTT GCAAAGTTTT CAACAAAAGT AAAGTTTGCT 2160GAAAGTTAGC AGTGTAACAT GTATTATCCT AACTTCTAAT CTTGTGGAAA TCAGACCCTA 2220TCAGTGCCCC TCAAAGCTGA AGTCCATCAG CATATGGCCA TACAACTAAT ACCCCTATTT 2280ATAGGGTTAG GAATGGCCAC TGCTACAGGA ATGGGAGTAA CAGGTTTATC TACTTCATTA 2340TCCTATTACC ACACACTCTT AAAGGATTTC TCAGACAGTT TACAAGAAAT AACAAAATCT 2400ATCCTTACTC TNTARTCCCA AATAGRTTCT TTGGCAGCAG TGACTCTC 2448 21 base pairsnucleotide single linear cDNA 54 CCTGAGTTCT TGCACTAACC C 21 23 basepairs nucleotide single linear cDNA 55 GTCCGTTGGG TTTCCTTACT CCT 23 1196base pairs nucleotide single linear cDNA 56 TTCCTGAGTT CTTGCACTAACCTCAAATGA GAGAAGTGCC GCCATAACTG CAACCCAAGA 60 GTTTGGCGAT CCCTGGTATCTCAGTCAGGT CAATGACAGG ATGACAACAG AGGAAAGATA 120 ATGATTCCCC ACAGGCCAGCAGGCAGTTCC CAGTGTAGAC CCTCATTAGG ACACAGAATC 180 AGAACATGGA GATTGGTGCCGCAGACATTT GCTAACTTGC GTGCTAGAAG GACTAAGGAA 240 AACTAGGAAG ATATGAATTATTCAATGATG TCCACTATAA CACAGGGGAA AGGAAGAAAA 300 TCCTACTGCC TTTCTGGAGAGACTAAGGGA GGCATTGAGG AAGCATACCA GGCAAGTGGA 360 CATTGGAGGC TCTGGAAAAGGGAAAAGTTG GGAAAAGTAT ATGTCTAATA GGGCTTGCTT 420 CCAGTGTGGT CTACAAGGACACTTTAAAAA AGATTGTCCA ATAGAAATAA GCCACCACCT 480 CGTCCATGCC CCTTATGTCAAGGGAATCAC TGGAAGGCCC ACTGCCCCAG GGGATGAAGG 540 TCCTCTGAGT CAGAAGCCACTAACCAGATG ATCCAGCAGC AGGACTGAGG GTGCCCGGGG 600 CAAGCGCCAG CCCATGCCATCACCCTCACA GAGCCCCAGG TATGCTTGAC CATTGAGGGT 660 CAGAAGGGTA CTGTCTCCTGGACACTGGCG GGCCTTCTCA GTCTTACTTT CCTGTCCTGG 720 ACAACTGTCC TCCAGATCTGTCACTGTCCG AGGGGTCCTA GGACAGCCAG TCACTAGATA 780 CTTCTCCCAG CCACTAAGTTGTGACTGGGG AACTTTACTC TTCCACATGC TTTTCTAATT 840 ATGCCTGAAA GCCCCACTCTCTTGTTAGGG GAGAGACATT CTAGCAAAAG CAGGGGCCAT 900 TATACATGTG AATATAGGAGAAGGAACAAC TGTTTGTTGT CCCCTGCTTG AGGAAGGAAT 960 TAATCCTGAA GTCCGGGCAACAGAAGGACA ATATGGACAA GCAAAGAATG CCCGTCCTGT 1020 TCAAGTTAAA CTAAAGGATTCCACCTCCTT TCCCTACCAA AGGCAGTACC CCCTCAGACC 1080 CGAGACCCAA CAAGAACTCCAAAAGATTGT AAAGGACCTA AAAGCCCAAG GCCTAGTAAA 1140 ACCAAGCAAT AGCCCTTGCAAGACTCCAAT TTTAGGAGTA AGGAAACCCA ACGGAC 1196 2391 base pairs nucleotidesingle linear cDNA 57 ATGATCCAGC AGCAGGACNG AGGGTGCCCG GGGCAAGCGCCAGCCCATGC CATCACCCTC 60 ACAGAGCCCC AGGTATGCTT GACCATTGAG GGTCAGAAGGGTNACTGTCT CCTGGACACT 120 GGCGGNGCCT TCTCAGTCTT ACTTTCCTGT CCTGGACAACTGTCCTCCAG ATCTGTCACT 180 GTCCGAGGGG TCCTAGGACA GCCAGTCACT AGATACTTCTCCCAGCCACT AAGTTGTGAC 240 TGGGGAACTT TACTCTTCCC ACATGCTTTT CTAATTATGCCTGAAAGCCC CACTCTCTTG 300 TTGGGGAGAG ACATTCTAGC AAAAGCAGGG GCCATTATACATGTGAATAT AGGAGAAGGA 360 ACAACTGTTT GTTGTCCCCT GCTTGAGGAA GGAATTAATCCTGAAGTCCG GGCAACAGAA 420 GGACAATATG GACAAGCAAA GAATGCCCGT CCTGTTCAAGTTAAACTAAA GGATTCCACC 480 TCCTTTCCCT ACCAAAGGCA GTACCCCCTC AGACCCGAGACCCAACAAGA ACTCCAAAAG 540 ATTGTAAAGG ACCTAAAAGC CCAAGGCCTA GTAAAACCAAGCAATAGCCC TTGCAAGACT 600 CCAATTTTAG GAGTAAGGAA ACCCAACGGA CAGTGGAGGTTAGTGCAAGA ACTCAGGATT 660 ATCAATGAGG CTGTTGTTCC TCTATACCCA GCTGTACCTAACCCTTATAC AGTGCTTTCC 720 CAAATACCAG AGGAAGCAGA GTGGTTTACA GTCCTGGACCTTAAGGATGC CTTTTTCTGC 780 ATCCCTGTAC GTCCTGACTC TCAATTCTTG TTTGCCTTTGAAGATCCTTT GAACCCAACG 840 TCTCAACTCA CCTGGACTGT TTTACCCCAA GGGTTCAGGGATAGCCCCCA TCTATTTGGC 900 CAGGCATTAG CCCAAGACTT GAGTCAATTC TCATACCTGGACACTCTTGT CCTTCAGTAC 960 ATGGATGATT TACTTTTAGT CGCCCGTTCA GAAACCTTGTGCCATCAAGC CACCCAAGAA 1020 CTCTTAACTT TCCTCACTAC CTGTGGCTAC AAGGTTTCCAAACCAAAGGC TCGGCTCTGC 1080 TCACAGGAGA TTAGATACTN AGGGCTAAAA TTATCCAAAGGCACCAGGGC CCTCAGTGAG 1140 GAACGTATCC AGCCTATACT GGCTTATCCT CATCCCAAAACCCTAAAGCA ACTAAGAGGG 1200 TTCCTTGGCA TAACAGGTTT CTGCCGAAAA CAGATTCCCAGGTACASCCC AATAGCCAGA 1260 CCATTATATA CACTAATTAN GGAAACTCAG AAAGCCAATACCTATTTAGT AAGATGGACA 1320 CCTACAGAAG TGGCTTTCCA GGCCCTAAAG AAGGCCCTAACCCAAGCCCC AGTGTTCAGC 1380 TTGCCAACAG GGCAAGATTT TTCTTTATAT GCCACAGAAAAAACAGGAAT AGCTCTAGGA 1440 GTCCTTACGC AGGTCTCAGG GATGAGCTTG CAACCCGTGGTATACCTGAG TAAGGAAATT 1500 GATGTAGTGG CAAAGGGTTG GCCTCATNGT TTATGGGTAATGGNGGCAGT AGCAGTCTNA 1560 GTATCTGAAG CAGTTAAAAT AATACAGGGA AGAGATCTTNCTGTGTGGAC ATCTCATGAT 1620 GTGAACGGCA TACTCACTGC TAAAGGAGAC TTGTGGTTGTCAGACAACCA TTTACTTAAN 1680 TATCAGGCTC TATTACTTGA AGAGCCAGTG CTGNGACTGCGCACTTGTGC AACTCTTAAA 1740 CCCAAACTTA TGCTGCCCAG AAGGATCTTT NTAGAGGTCCCCTTAGCCAA CCCTGACCTC 1800 AACTATATAT ATACTGATGG AAGTTCGTTT GTAGAAAAGGGATTACAAAG GGNAGGATAT 1860 NCCATAGGTG TTAGTGATAA AGCAGTACTT GAAAGTAAGCCTCTTCCCCC CCAGGGACCA 1920 GCGCCCCCGT TAGCAGAACT AGTGGCACTG ACCCCGCGAGCCTTAGAACT TTGGAAAGGG 1980 AGGAGGATAA ATGTGTATAC AGATAGCAAG TATGCTTATCTAATCCGAAA TGCCCATGTT 2040 GTTTATCTAA TCCGAAATGC CCATGTTGCA ATATGGAAAGAAAGGGAGTT CCTAACCTCT 2100 GGGGGAACCC CCATTAAATA CCACAAGTTA ATCATGGAGTTATTGCACAC AGTGCAAAAA 2160 CTCAAGGAGG TGGAAGTCTT ACACTGCCAA AGCCATCAGAAAAGGGAAAG GGGAGAAGAG 2220 CAGCATAAGT GGCTACAGAG GCAAGGAAAG ACTAGCAGAAAGGAAAGAGA GAAAGAGACA 2280 GAAAGTCAGA GAGAGAGAGA GGAAGAGACA GAGCACAAAGAGGGAGTCAG AGAGAGAGAG 2340 AGACAGAGAG TCAGAGAGAA GGAAAGAGAG AGAGGAAGAGACAAAGAATG A 2391 1722 base pairs nucleotide single linear cDNA 58TGGAGAATAG CAGCATAAGT TGGCTGGCAG AAGTAGGGAA AGACAGCAAG AAGTAAAGAA 60AAAAARGAGA AAGTCAGAGA AAGAAAAAAA GAGAGGAAGA AACAAAGAAG AACTTGAAGA 120GAGAAAGAAG TAGTAAAGAA AAAACAGTAT ACCCTATTCC TTTAAAAGCC AGGGTAAATT 180TCTGTCTACC TAGCCAAGGC ATATTCTTCT TATGTGGAAC ATCAACCTAT ATCTGCCTCC 240CCACTAACTG GACAGGCACC TGAACCTTAG TCTTTCTAAG TCCCAACATT AACATTGCCC 300CAGGAAATCA GACCCTATTG GTACCTGTCA AAGCTAAAGT CCCGTCAGTG CAGAGCCATA 360CAACTAATAT CCCTATTTAT AGGGTTAGGA ATGGCTACTG CTACAGGAAC TGGAATAGCC 420GGTTTATCTA CTTCATTATC CTACTACCAT ACACTCTCAA AGAATTTCTC AGACAGTTTG 480CAAGAAATAA TGAAATCTAT TCTTACTTTA CAATCCCAAT TAGACTCTTT GGCAGCAATG 540ACTCTCCAAA ACCGCCGAGG CCCACACCTC CTCACTGCTG AGAAAGGAGG ACTCTGCACC 600TTCTTAGGGG AAGAGTGTTG TTTTTACACT AACCAGTCAG GGATAGTACG AGATGCCACC 660TGGCATTTAC AGGAAAGGGC TTCTGATATC AGACAATGCC TTTCAAACTC TTATACCAAC 720CTCTGGAGTT GGGCAACATG GCTTCTTCCA TTTCTAGGTC CCATGGCAGC CATCTTGCTG 780TTACTCACCT TTGGGCCCTG TATTTTTAAG CTTCTTGTCA AATTTGTTTC CTCTAGGATC 840GAAGCCATCA AGCTACAGAT GGTCTTACAA ATGGAACCCC AAATGAGTTC AACTAACAAC 900TTCTACCAAG GACCCCTGGA ACGATCCACT GGCACTTCCA CTAGCCTAGA GATTCCCCTC 960TGGAAGACAC TACAACTGCA GGGCCCCTTC TTTGCCCCTA TCCAGCAGGA AGTAGCTAGA 1020GCGGTCATCG GCCAAATTCC CAACAGCAGT TGGGGTGTCC TGTTTAGAGG GGGGATTGAA 1080GAGGTGACAG CCTGCTGGCA GCCTCACAGC CCTCGTTGGY TCTCAGTGCC TCCTCAGCCT 1140TGGTGCCCAC TCTGGCCGTG CTTGAGGAGC CCTTCAGCCT GCCACTGCAC TGTGGGAGCC 1200TCTTTCTGGG CTGGACAAGG CCGGAGCCAG CTCCCTCAGC TTGCAGGGAG GTATGGAGGG 1260AGAGATGCAG GCGGGAACCA GGGCTGCGCA TGGCGCTTGC GGGCCAGCAT GAGTTCCAGG 1320TGGGCGTGGG CTCGGCGGGC CCCACACTCG GGCAGTGAGG GGCTTAGCAC CTGGGCCAGA 1380CAGATGCTGT GCTCAACTTC TTCGCTGGGC CTTAGCTGCC TTCCCCGTGG GGCAGGGCTY 1440CGGGAACMTG CAGCCTGCCC ATGCTTGAGC CCCCCACCCC GCCGTGGGTT CYTGCACAGC 1500CCAAGCTTCC CGGACAAGCA CCACCCCTTA TCCACGGTGC CCAGTCCCAT CAACCACCCA 1560AGGGTTGAGG AGTGCGGGCA CACAGCGCGG GATTGGCAGG CAGTTCCACT TGCGGCCTTG 1620GTGCGGGATC CACTGCGTGA AGCCAGCTGG GCTCCTGAGT CTGGTGGGGA CTTGGAGAAT 1680CTTTATGTCT AGCTAAGGGA TTGTAAATAC ACCAATCAGC AC 1722 495 base pairsnucleotide single linear cDNA 59 CTTCCCCAAC TAATAAGGAC CCCCCTTTCAACCCAAACAG TCCAAAAGGA CATAGACAAA 60 GGAGTAAACA ATGAACCAAA GAGTGCCAATATTCCCTGGT TATGCACCCT CCAAGCGGTG 120 GGAGAAGAAT TCGGCCCAGC CAGAGTGCATGTACCTTTTT CTCTCTCACA CTTGAAGCAA 180 ATTAAAATAG ACNTAGGTNA ATTNTCAGATAGCCCTGATG GYTATATTGA TGTTTTACAA 240 GGATTAGGAC AATCCTTTGA TCTGACATGGAGAGATATAA TATTACTGCT AAATCAGACG 300 CTAACCTCAA ATGAGAGAAG TGCTGCCATAACTGGAGCCC GAGAGTTTGG CAATCTCTGG 360 TATCTCAGTC AGGTCAATGA TAGGATGACAACGGAGGAAA GAGAACGATT CCCCACAGGG 420 CAGCAGGCAG TTCCCAGTGT AGCTCCTCATTGGGACACAG AATCAGAACA TGGAGATTGG 480 TGCCGCAGAC ATTTA 495 2503 basepairs nucleotide single linear cDNA 60 CCAAGAACCC ACCAATTCCG GANCACATTTTGGCGACCAC GAAGGGACTT TCGCATATCG 60 CCAAGCGGTG AGACAATAGC CGAGCGGTGAGACCTTTCCC AATCGCCAAG CAGTGAGTAC 120 CATCAGACCC CTTTCACTTG CTATTCTGTCCTATCTTTCT TTAGAATTCG GGGGCTAAAT 180 ACCGGGCATC TGTCAGCCAT TTAAAAGTGACTAGCGGGCC GCCGGACTAA AGACACGGGT 240 GTCAAGCTTT CTGGGAAAGG GCTCTCTAACAACCCCCAAC TCTTTGGAGT TGGGACCGTT 300 GGTTTGCCTA GAACCAGCTT CCGCTTTTCCTGTACTTCTG GGCTGAGCCG TGGGTTGACA 360 GTGAAGGAAA GCCATGCATC TCCGGGGTCTCGMCAACATG TTGGTTGACC CTGCGGCCAT 420 GAGTGGAACT CTCAAAAGCA TGTCGCCCAAGCGACACTCG CCTATCTATC CTATCTATCC 480 TGACCCTTGC CCTCTGGGTC CTAATGCCTGCCAGACAAAC TTCCTCTCGC CTCTCTTCTC 540 TGAAGCTAGA ACCGCTTCTA AAAATTGCTACCTGGTCTCT GGTGCTTTTC CTARTTTCTC 600 CTATAAAGAA TGAWTTCTAG TATTAAACTCCAGGACTCTG TTACCTTCTT TAGGCACCCG 660 GGCTCACCAA TCAGAAAGAC ACAGTTTTTGCCCAAGGCCC CATCGTAGTG GGGACTACCT 720 GGAATTTTAG GATCCCTCCT CAGACTAACAGGCCTAACAA AAGTTATTCC TGAAGCTAGG 780 ATATGGGGAG CCTCAGAAAT TGTATCCCTCCTATTCATAT AAGTGAGAAC AAAAGGTGTC 840 ACTCTTCCAA CCCTGAAGAT CCCCTCCCTCCCTCAGGGTA TGGCCCTCCA TTTCATTTTT 900 GTGGCATAAC ATCTTTATAG GATGGGGTAAAGTCCCAATA CTAACAGGAG AATGCTTAGG 960 ACTCTAACAG GTTTTTGAGA ATGCGTCAGTAAGGGCCACT AAATCTGATT TTTCTCAGTC 1020 GGTCCTCCTT GTGGTCTAGG AGGACAGGCAAGGTTGTGCA GGTTTTCGAG AATGCGTCAG 1080 TAAGGACCAC TAAATCCGAC CTTCCTCGGTCCTCCATGTG GTCTGGGAGG AAAACTAGTG 1140 TTTCTGCTGC TGCGTCGGTG AGCGCAACTATTCAAGTCAG CAGGGTCCAG GGACCGTTGC 1200 AGGTTCTTGG GCAGGGGTTG TTTCTGCTGCTGCATTGGTG AATGCAACTA TTCTGATCAG 1260 CAGGGTCCCA GGACCATTGC AGGTCCTTGGGCAGGGAGAG AAACAAAACA AACCAAAACT 1320 GTGGGCGGTT TTGTCTTTCA TATGGGAAACACTCAGGCAT CAACAGGTTC ACCCTTGAAA 1380 TGCATCCTAA GCCATTGGGA CCAATTTGACCCACAAACCC TGAAAAAGAG GAGGCTCATT 1440 TTTTCCTGCA CTACGGCTTG GCCCCAATATTCTCTTTYTG ATGGGGAAAA ATGGCCACCT 1500 GAGGGAAGCA CAAATTACAA TAYTATCCTACAGCYTGATC TTTTCTGTAA GAGGGAAGGC 1560 AAATGGAGTG AATACCTTAT GTCCAAGCTTTCTTTTCATT GAGGGAGAAT ACACAACTAT 1620 GCAAAGCTTG CAATTTACAT CCCACAGGAGGACCCTTCAG CTTACCCCCA TATCCTAGCC 1680 TCCCTATAGC TTCCCTTCCT ATTGATGATACTCCTCCTCT AATCTCCCCT GCCCAGAAGG 1740 AAATAAGCAA AGAAATCTCC AAAGGTCCACAAAAACCCCC GGGCTATCGG TTATGTCCCT 1800 TCAAGYTGTA GGGGGAGGGG AATTTGGCCCAACCCGGGTG CATGTCCCTT CTCCCTCTCT 1860 GATTTAAAGC AGATCAAGGC AGACCTGGGGAAGTTTTCAG ATGATCCTGA TAGGTACATA 1920 GATGTCCTAC AGGGTCTAGG GCAAACCTTTGACCTCACTT GGAGAGACGT CATGCTACTG 1980 TTAGATCAAA CCCTGGCCTT TAATGAAAAGAATGCGGCTT TAGCTGCAGC CTGAGAGTTT 2040 GGAGATACCT GGTATCCTAG TCAAGTAAATGAAAGAATGA CAGCCGAAGA AAGGGACAAC 2100 TTCCTTACTG GTCAGCAACC CATCCCCAGTATGGATCCCC ACTGGGACTT TGACTCAGAT 2160 CATGGGGACT GGAGTCGTAA ACATCTGTTGATCTGTGTTC TGGAAGGACT AAGGAGAATT 2220 GGGAAAAAGC CCATGAATTA TTCAATGATATCCACCATAA CCCAGGGAAA GGAAGAAAAT 2280 CCTTCTGCCT TCCTCGAGCG GCTACAAGAGGCCTTAAGAA AATATACTCC CCTGTCACCC 2340 GAATCACTCG AGGGTCAATT GATTCTAAAAGATAAGTTTA TTACCCAATC AGCCACAGAT 2400 ATCAGGAGAA AGCTCCAAAA GCAAGCCCTGAGCCTGAACA AAATCTAGAG ACATTATTAA 2460 ACCTGGCAAC CTTGGTGTTC TATAATAGGGACCAAGAGGA ACA 2503 1167 base pairs nucleotide single linear cDNA 61AAGGAAACTC AGAAAGCCAA TACCCATTTA GTAAGATGGA CACCAGAAGC AGAAGCAGCT 60TTCCAGGCCC TAAAGAAATC CCTAACCCAA GCCCCAGTGT TAAGCTTGCC AACGGGGCAA 120GACTTTTCTT TATATGTCAC AGAAAAACAG GAATAGCTCT AGGAGTCCTT ACACAGGTCC 180AAGGGACAAG CTTGCAACCT GTGGCATACC TGAGTAAGGA AACTGATGTA NTGGCAAAGG 240GTTGGCCTCA TTGTTTACAG GTAGGGCAGC AGTAGCAGTC TTAGTTTCTG AAACAGTTAA 300AATAATACAG GGAAGAGATC TTACTGTGTG GACATCTCAT GATGTGAACG GCATACTCAC 360TGCTAAAGAG GACTTGTGGC TGTCAGACAA CCATTTACTT AAATAGCAGG TTCTATTACT 420TGAAGTGCCA GTGCTGCGAC TGCACATTTG TGCAACTCTT AACCCAGCCA CATTTCTTCC 480AGACAATGAA GAAAAGATAG AACATAACTG TCAACAAGTA ATTGCTCAAA CCTATGCTGC 540TCGAGGGGAC CTTCTAGAGG TTCCCTTGAC TGATCCCGAC CTCAACTTGT ATACTGATGG 600AAGTTCCTTG GCAGAAAAAG GACTTTGAAA AGCGGGGTAT GCAGTGATCA GTGATAATGG 660AATACTTGAA AGTAATCGCC TCACTCCAGG AACTAGTGCT CACCTGGCAG AACTAATAGC 720CCTCACTTGG GCACTAGAAT TAGGAGAAGG AAAAAGGGTA AATATATATT CAGACTCTAA 780GTATGCTTAC CTAGTCCTCC ATGCCCATGC AGCAATATGG AGAGAGAGGG AATTCCTAAC 840TTCTGAGGGA ACACCTATCA ACCATCAGGG AAGCCATTAG GAGATTATTA TTGGCTGTAC 900AGAAACCTAA AGAGGTGGCA GTCTTACACT GCCAGGGTCA TCAGGAAGAA GAGGAAAGGG 960AAATAGAAGG CAATCGCCAA GCGGATATTG AAGCAAAAAA AGCCGCAAGG CAGGACTCTC 1020CATTAGAAAT GCTTATAGAA GGACCCCTAG TATGGGGTAA TCCCCTCTGG GAAACCAAGC 1080CCCAGTACTC AGCAGGAAAA ATAGAATAGG AAACCTCACA AGGACATACT TTCCTCCCCT 1140CCAGATGGCT AGCCACTGAG GAAGGAA 1167 78 base pairs nucleotide singlelinear cDNA 62 TCCAAAGGCA CCAGGGCCCT CAGTGAGGAA CGTATCCAGC CTATACTGGCTTATCCTCAT 60 CCCAAAACCC TAAAGCAA 78 26 amino acids amino acid linearpeptide 63 Ser Lys Gly Thr Arg Ala Leu Ser Glu Glu Arg Ile Gln Pro IleLeu 1 5 10 15 Ala Tyr Pro His Pro Lys Thr Leu Lys Gln 20 25 28 basepairs nucleotide single linear cDNA 64 AAATGTCTGC GGCACCAATC TCCATGTT 2830 base pairs nucleotide single linear cDNA 65 AAGGGGCATG GACGAGGTGGTGGCTTATTT 30 21 base pairs nucleotide single linear cDNA 66 GGAGAAGAGCAGCATAAGTG G 21 25 base pairs nucleotide single linear cDNA 67GTGCTGATTG GTGTATTTAC AATCC 25 34 base pairs nucleotide single linearcDNA 68 GACTCGCTGC AGATCGATTT TTTTTTTTTT TTTT 34 30 base pairsnucleotide single linear cDNA 69 GCCATCAAGC CACCCAAGAA CTCTTAACTT 30 30base pairs nucleotide single linear cDNA 70 CCAATAGCCA GACCATTATATACACTAATT 30 23 base pairs nucleotide single linear cDNA 71 GCCATAACTGCAACCCAAGA GTT 23 23 base pairs nucleotide single linear cDNA 72GGACGAGGTG GTGGCTTATT TCT 23 25 base pairs nucleotide single linear cDNA73 AACTTGCGTG CTAGAAGGAC TAAGG 25 24 base pairs nucleotide single linearcDNA 74 AACTTTTCCC TTTTCCAGAT CCTC 24 22 base pairs nucleotide singlelinear cDNA 75 GCATACCAGG CAAGTGGACA TT 22 25 base pairs nucleotidesingle linear cDNA 76 CTGTCCGTTG GGTTTCCTTA CTCCT 25 24 base pairsnucleotide single linear cDNA 77 GAGGCTCTGG AAAAGGGAAA AGTT 24 25 basepairs nucleotide single linear cDNA 78 CTGTCCGTTG GGTTTCCTTA CTCCT 25 25base pairs nucleotide single linear cDNA 79 AGGAGTAAGG AAACCCAACG GACAG25 25 base pairs nucleotide single linear cDNA 80 TGTATATAAT GGTCTGGCTATTGGG 25 25 base pairs nucleotide single linear cDNA 81 AGGAGTAAGGAAACCCAACG GACAG 25 26 base pairs nucleotide single linear cDNA 82TTCGGCAGAA ACCTGTTATG CCAAGG 26 22 base pairs nucleotide single linearcDNA 83 CTCGATTTCT TGCTGGGCCT TA 22 20 base pairs nucleotide singlelinear cDNA 84 GTTGATTCCC TCCTCAAGCA 20 20 base pairs nucleotide singlelinear cDNA 85 CTCTACCAAT CAGCATGTGG 20 19 base pairs nucleotide singlelinear cDNA 86 TGTTCCTCTT GGTCCCTAT 19 433 amino acids amino acid linearpeptide 87 Met Ala Thr Ala Thr Gly Thr Gly Ile Ala Gly Leu Ser Thr SerLeu 1 5 10 15 Ser Tyr Tyr His Thr Leu Ser Lys Asn Phe Ser Asp Ser LeuGln Glu 20 25 30 Ile Met Lys Ser Ile Leu Thr Leu Gln Ser Gln Leu Asp SerLeu Ala 35 40 45 Ala Met Thr Leu Gln Asn Arg Arg Gly Pro His Leu Leu ThrAla Glu 50 55 60 Lys Gly Gly Leu Cys Thr Phe Leu Gly Glu Glu Cys Cys PheTyr Thr 65 70 75 80 Asn Gln Ser Gly Ile Val Arg Asp Ala Thr Trp His LeuGln Glu Arg 85 90 95 Ala Ser Asp Ile Arg Gln Cys Leu Ser Asn Ser Tyr ThrAsn Leu Trp 100 105 110 Ser Trp Ala Thr Trp Leu Leu Pro Phe Leu Gly ProMet Ala Ala Ile 115 120 125 Leu Leu Leu Leu Thr Phe Gly Pro Cys Ile PheLys Leu Leu Val Lys 130 135 140 Phe Val Ser Ser Arg Ile Glu Ala Ile LysLeu Gln Met Val Leu Gln 145 150 155 160 Met Glu Pro Gln Met Ser Ser ThrAsn Asn Phe Tyr Gln Gly Pro Leu 165 170 175 Glu Arg Ser Thr Gly Thr SerThr Ser Leu Glu Ile Pro Leu Trp Lys 180 185 190 Thr Leu Gln Leu Gln GlyPro Phe Phe Ala Pro Ile Gln Gln Glu Val 195 200 205 Ala Arg Ala Val IleGly Gln Ile Pro Asn Ser Ser Trp Gly Val Leu 210 215 220 Phe Arg Gly GlyIle Glu Glu Val Thr Ala Cys Trp Gln Pro His Ser 225 230 235 240 Pro ArgTrp Xaa Ser Val Pro Pro Gln Pro Trp Cys Pro Leu Trp Pro 245 250 255 CysLeu Arg Ser Pro Ser Ala Cys His Cys Thr Val Gly Ala Ser Phe 260 265 270Trp Ala Gly Gln Gly Arg Ser Gln Leu Pro Gln Leu Ala Gly Arg Tyr 275 280285 Gly Gly Arg Asp Ala Gly Gly Asn Gln Gly Cys Ala Trp Arg Leu Arg 290295 300 Ala Ser Met Ser Ser Arg Trp Ala Trp Ala Arg Arg Ala Pro His Ser305 310 315 320 Gly Ser Glu Gly Leu Ser Thr Trp Ala Arg Gln Met Leu CysSer Thr 325 330 335 Ser Ser Leu Gly Leu Ser Cys Leu Pro Arg Gly Ala GlyLeu Arg Glu 340 345 350 Xaa Ala Ala Cys Pro Cys Leu Ser Pro Pro Pro ArgArg Gly Phe Leu 355 360 365 His Ser Pro Ser Phe Pro Asp Lys His His ProLeu Ser Thr Val Pro 370 375 380 Ser Pro Ile Asn His Pro Arg Val Glu GluCys Gly His Thr Ala Arg 385 390 395 400 Asp Trp Gln Ala Val Pro Leu AlaAla Leu Val Arg Asp Pro Leu Arg 405 410 415 Glu Ala Ser Trp Ala Pro GluSer Gly Gly Asp Leu Glu Asn Leu Tyr 420 425 430 Val 433 693 base pairsnucleotide single linear cDNA 88 CTTCCCCAAC TAATAAGGAC CCCCCTTTCAACCCAAACAG TCCAAAAGGA CATAGACAAA 60 GGAGTAAACA ATGAACCAAA GAGTGCCAATATTCCCTGGT TATGCACCCT CCAAGCGGTG 120 GGAGAAGAAT TCGGCCCAGC CAGAGTGCATGTACCTTTTT CTCTCTCACA CTTGAAGCAA 180 ATTAAAATAG ACNTAGGTNA ATTNTCAGATAGCCCTGATG GYTATATTGA TGTTTTACAA 240 GGATTAGGAC AATCCTTTGA TCTGACATGGAGAGATATAA TATTACTGCT AAATCAGACG 300 CTAACCTCAA ATGAGAGAAG TGCTGCCATAACTGGAGCCC GAGAGTTTGG CAATCTCTGG 360 TATCTCAGTC AGGTCAATGA TAGGATGACAACGGAGGAAA GAGAACGATT CCCCACAGGG 420 CAGCAGGCAG TTCCCAGTGT AGCTCCTCATTGGGACACAG AATCAGAACA TGGAGATTGG 480 TGCCGCAGAC ATTTACTAAC TTGCGTGCTAGAAGGACTAA GGAAAACTAG GAAGACTATG 540 AATTATTCAA TGATGTCCAC TATAACACAGGGGAAAGGAA GAAAATCCTA CTGCCTTTCT 600 GGAGAGACTA AGGGAGGCAT TGAGGAAGCATACCAGGCAA GTGGACATTG GAGGCTCTGG 660 AAAAGGGAAA AGTTGGGCAA ATTGAATGCCTAA 693 1577 base pairs nucleotide single linear cDNA 89 AACTTGCGTGCTAGAAGGAC TAAGGAAAAC TAGGAAGACT ATGAATTATT CAATGATGTC 60 CACTATAACACAGGGGAAAG GAAGAAAATC CTACTGCCTT TCTGGAGAGA CTAAGGGAGG 120 CATTGAGGAAGCATACCAGG CAAGTGGACA TTGGAGGCTC TGGAAAAGGG AAAAGTTGGG 180 CAAATTGAATGCCTAATAGG GCTTGCTTCC AGTGCAGTCT ACAAGGACGC TTTAGAAAAG 240 ATTGTCCAAGTAGAAATAAG CCGCCCCTCG TCCATGCCCC TTATGTCAAG GGAATCACTG 300 GAAGGCCTACTGCCCCAGGG GACGAAGGTC CTCTGAGTCA GAAGCCACTA ACCTGATGAT 360 CCAGCAGCAGGACTGAGGGT GCCCGGGGCA AGTGCCAGCC CATGCCATCA CCCTCAGAGC 420 CCCGGGTATGTTTGACCATT GAGAGCCAGG AAGTTAACTG TCTCCTGGAC ACTGGCGCAG 480 CCTTCTCAGTCTTACTTTCC TGTCCCAGAC AATTGTCCTC CAGATCTGTC ACTATCCGAG 540 GGGTCCTAAGACAGCCAGTC ACTACATACT TCTCTCAGCC ACTAAGTTGT GACTGGGGAA 600 CTTTACTCTTTTCACATGCT TTTCTAATTA TGCCTGAAAG CCCCACTCCC TTGTTAGGGA 660 GAGACATTTTAGCAAAAGCA GGGGCCATTA TACACCTGAA CATAGGAAAA GGAATACCCA 720 TTTGCTGTCCCCTGCTTGAG GAAGGAATTA ATCCTGAAGT CTGGGCAATA GAAGGACAAT 780 ATGGACAAGCAAAGAATGCC CGTCCTGTTC AAGTTAAACT AAAGGATTCT GCCTCCTTTC 840 CCTACCAAAGGAAGTACCCT CTTAGACCCG AGGCCCTACA AGGACTCAAA AGATTGTTAA 900 GGACCTAAAAGCCCAAGGCC TAGTAAAACC ATGCAGTAGC CCCTGCAATA CTCCAATTTT 960 AGGAGTAAGGAAACCCAACG GACAGTGGAG GTTAGTGCAA GATCTCAGGA TTATTAATGA 1020 GGCTGTTTTTCCTCTATACC CAGCTGTATC TAGCCCTTAT ACTCTGCTTT CCCTAATACC 1080 AGAGGAAGCAGAGTAGTTTA CAGTCCTGGA CCTTAAGGAT GCCTCTTTCT GCATCCCTGT 1140 ACATCCTGATTCTCAATTCT TGTTTGTCTT TGAAGATCCT TTGAACCCAA TGTCTCAATT 1200 CACCTGGACTGTTTTACCCC AGGGGTTCCG GGATAGCCCC CATCTATTTG GCCAGGCATT 1260 AGCCCAAGACTTGAGCCAAT TCTCATACCT GGACATCTTG TCCTTCGGTA TGGGATGATT 1320 TAATTTTAGCCACCCGTTCA GAAACCTTGT GCCATCAAGC CACCCAAGCG TTCTTAAATT 1380 TCCTCACTCCGTGTGGCTAC AAGGTTTCCA AACCAAAGGC TCAGCTCTGC TCACAGCAGG 1440 TTAAATACTTAGGGTTAAAA TTATCCAAAG GCACCAGGGC CCTCTGTGAG GAATGTATCC 1500 AACCTGTACTGGCTTATCTT CATCCCAAAA CCCTAAAGCA ACTAAGAAGG TCCTTGGCAT 1560 AACAGGTTTCTGCCGAA 1577 182 amino acids amino acid linear peptide 90 Ser Ser SerArg Thr Glu Gly Ala Arg Gly Lys Cys Gln Pro Met Pro 1 5 10 15 Ser ProSer Glu Pro Arg Val Cys Leu Thr Ile Glu Ser Gln Glu Val 20 25 30 Asn CysLeu Leu Asp Thr Gly Ala Ala Phe Ser Val Leu Leu Ser Cys 35 40 45 Pro ArgGln Leu Ser Ser Arg Ser Val Thr Ile Arg Gly Val Leu Arg 50 55 60 Gln ProVal Thr Thr Tyr Phe Ser Gln Pro Leu Ser Cys Asp Trp Gly 65 70 75 80 ThrLeu Leu Phe Ser His Ala Phe Leu Ile Met Pro Glu Ser Pro Thr 85 90 95 ProLeu Leu Gly Arg Asp Ile Leu Ala Lys Ala Gly Ala Ile Ile His 100 105 110Leu Asn Ile Gly Lys Gly Ile Pro Ile Cys Cys Pro Leu Leu Glu Glu 115 120125 Gly Ile Asn Pro Glu Val Trp Ala Ile Glu Gly Gln Tyr Gly Gln Ala 130135 140 Lys Asn Ala Arg Pro Val Gln Val Lys Leu Lys Asp Ser Ala Ser Phe145 150 155 160 Pro Tyr Gln Arg Lys Tyr Pro Leu Arg Pro Glu Ala Leu GlnGly Leu 165 170 175 Lys Arg Leu Leu Arg Thr 180 36 base pairs nucleotidesingle linear cDNA 91 AGATCTGCAG AATTCGATAT CACCCCCCCC CCCCCC 36 22 basepairs nucleotide single linear cDNA 92 AGATCTGCAG AATTCGATAT CA 22

What is claimed is:
 1. A process for detecting a pathological and/orinfective agent associated with multiple sclerosis and/or rheumatoidarthritis, in a biological sample, comprising bringing a nucleic acidinto contact with the biological sample, and detecting hybridization,said nucleic acid comprising a nucleotide sequence having a successionof at least 100 contiguous monomers of a nucleotide sequence selectedfrom the group consisting of said sequences SEQ ID NO: 46, SEQ ID NO:51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 56, SEQ ID NO: 58, SEQ IDNO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 89 and theircomplementary sequences.
 2. A process for detecting a pathologicaland/or infective agent associated with multiple sclerosis and/orrheumatoid arthritis, in a biological sample, comprising bringing anucleic acid into contact with the biological sample, and detectinghybridization, said nucleic acid consisting of 100 or more contiguousmonomers of a nucleotide sequence selected from the group consisting ofSEQ ID NO: 46, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO:56, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ IDNO: 89 and their complementary sequences.
 3. A process for detecting apathological and/or infective agent associated with multiple sclerosisand/or rheumatoid arthritis, in a biological sample, comprising bringinga nucleic acid into contact with the biological sample, and detectinghybridization, said nucleic acid comprising 100 or more contiguousmonomers of a nucleotide sequence selected from the group consisting ofSEQ ID NO: 46, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO:56, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ IDNO: 89 and their complementary sequences.
 4. A probe for specificallyhybridizing with a nucleic acid of a virus associated with multiplesclerosis, said probe comprising at least ten contiguous monomers of thenucleotide sequence of a nucleic acid, said nucleic acid comprising anucleotide sequence having a succession of at least 100 contiguousmonomers of a nucleotide sequence selected from the group consisting ofSEQ ID NO: 46, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO:56, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ IDNO: 89 and their complementary sequences.
 5. A probe as defined in claim4, having 10 to 100 contiguous monomers of said nucleotide sequence ofthe nucleic acid.
 6. A specific primer for amplification bypolymerization of a nucleic acid of a virus associated with multiplesclerosis, said primer comprising at least ten contiguous monomers ofthe nucleotide sequence of a nucleic acid, said nucleic acid comprisinga nucleotide sequence having a succession of at least 100 contiguousmonomers of a nucleotide sequence selected from the group consisting ofSEQ ID NO: 46, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO:56, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ IDNO: 89 and their complementary sequences.
 7. A primer as defined inclaim 6, having 10 to 100 contiguous monomers of said nucleotidesequence of the nucleic acid.
 8. A process for detecting a pathologicaland/or infective agent associated with multiple sclerosis, in abiological sample, comprising bringing a probe according to claim 4 intocontact with the biological sample, and detecting hybridization.